pVAX1/SjscFv-IL18对日本血吸虫病肝纤维化的影响及其作用机制的研究
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摘要
研究背景
血吸虫病(schistosomiasis)是一种流行广泛、严重危害人类健康和社会经济发展的人兽共患寄生虫病,我国是日本血吸虫病(schistosomiasis japonica)流行最严重的四个国家之一。日本血吸虫(Schistosoma japonicum, Sj)致病的主要原因是雌虫产出的大量虫卵沉积于肝、肠等组织中,形成虫卵肉芽肿、组织损伤和继发性肝纤维化。肝纤维化的进一步发展导致肝硬化,患者可因上消化道出血、肝性昏迷等严重并发症而致死。因此,阻止虫卵肉芽肿病变的形成,对于控制日本血吸虫病的进程、防止晚期血吸虫病的发生具有重要的意义。
目前,临床上采用单一的吡喹酮治疗血吸虫病仍存在一定缺陷,吡喹酮只能杀灭童虫和成虫,而不能阻止肝组织内的虫卵继续分泌抗原,虫卵可溶性抗原(soluble matured egg antigen, SEA)可引起组织内虫卵肉芽肿反应,由于血吸虫病患者发现时多己转为慢性,肝组织内虫卵已经或正在形成虫卵肉芽肿或肝纤维化,有效杀虫治疗后病人依然有转化为晚期的可能,SEA诱导产生的免疫病理反应并不因杀虫治疗而终止。IL-18是目前己知的对血吸虫病肝纤维化具有治疗作用的细胞因子,然而单纯使用细胞因子治疗需连续、大剂量的用药,势必导致毒副作用的增加以及宿主体内细胞因子的严重失衡。
因此,我们拟利用实验室前期筛选得到的对日本血吸虫雌虫、未成熟虫卵、成熟虫卵等多个阶段均具有高特异性免疫靶向作用及识别能力的单链抗体(Sj single-chain Fv antibody, SjscFv),将其与对血吸虫性肝纤维化具有治疗作用的细胞因子IL-18融合表达,产生SjscFv-IL18融合蛋白,借助SjscFv的免疫及靶向作用携带IL-18进入虫卵肉芽肿和肝纤维化形成的局部,特异性提高局部免疫作用及IL-18浓度,诱导日本血吸虫感染慢性期从Th2型免疫应答转化为Th1型优势的免疫应答,同时能够极大地降低IL-18对其它正常组织器官的毒副作用,以期达到更好的抗日本血吸虫性病纤维化的效果。
研究目的
构建真核重组质粒pVAX1/SjscFv-IL18,转染巨噬细胞后,使用SEA刺激后的巨噬细胞上清液处理肝星状细胞(HSCs),观察对HSCs激活、增殖和凋亡的影响,并检测HSCs中与细胞外基质(ECM)沉积相关的基因表达,评估其抗肝纤维化的能力；并在日本血吸虫病肝纤维化小鼠中研究其对虫卵肉芽肿及肝纤维化形成的影响及其作用机制。
研究方法
1、真核重组质粒pVAX1/SjscFv-IL18的构建；
采用SOE-PCR技术构建重组质粒pVAX1/SjscFv-IL18和pVAX1/S/scFv,转化大肠杆菌后,进行大量表达,并通过双酶切和测序鉴定,使用生物信息学方法分析其结构特征。
2、pVAX1/SjscFv-IL18对HSCs胶原生成与降解的影响
将各组重组真核质粒转染巨噬细胞后,采用SEA刺激巨噬细胞后的上清液处理HSCs,应用实时荧光定量PCR(qPCR)检测胶原相关基因mRNA水平、MTT检测HSCs细胞增殖、流式细胞术检测HSCs凋亡。
3、pVAX1/SjscFv-IL18对日本血吸虫病肝纤维化的影响
建立日本血吸虫病肝纤维化小鼠模型,将pVAX1/SjscFv-IL18注入小鼠体内,采用]HE、Masson染色方法分别检测肝组织内虫卵肉芽肿的大小、肝组织内胶原沉积的情况,应用ELISA检测小鼠Thl/Th2型细胞因子、IL-18,免疫组化检测肝组织中TGF β1和IL-18, qPCR检测肝脏中胶原相关基因mRNA水平。
研究结果
1、PCR、双酶切及测序结果显示重组质粒插入序列与目的序列一致,CD-Search证实所构建质粒表达蛋白中包含目的蛋白所需的两个Ig超家族和IL-1超家族保守结构域,说明真核重组质粒pVAX1/SjscFv-IL18构建成功。
2. pVAX1/SjscFv-IL18转染巨噬细胞后,采用SEA刺激该巨噬细胞产生的上清液处理HSCs,与对照组相比,能够部分降低HSCs中a-SMA的mRNA水平,并抑制HSCs增殖、促进HSCs凋亡,上调MMPs-1的同时下调Col Ⅰ、Col Ⅲ和TIMP-1的mRNA表达。
3、pVAX1/SjscFv-IL18注入日本血吸虫病肝纤维化小鼠后,能够降低肝组织内虫卵肉芽肿的大小,减少肝组织内胶原的沉积,并在体内表达大量的IL18,诱导Thl型优势的免疫反应,减少肝脏中TGF β1表达,上调MMPs-1的同时下调Col Ⅰ、Col Ⅲ和TIMP-1的mRNA表达,且能够在虫卵肉芽肿局部提高IL-18的浓度,从而产生更好的抗日本血吸虫病肝纤维化的作用。
结论
1、成功构建了真核重组质粒pVAX1/SjscFv-IL18。
2、pVAX1/SjscFv-IL18在体外能够通过影响巨噬细胞内细胞因子的分泌进而抑制HSCs中胶原的生成,促进胶原降解。
3、pVAX1/SjscFv-IL18能够通过SjscFv的免疫靶向作用提高IL-18的生物学活性,从而抑制虫卵肉芽肿的形成,并产生更好的抗日本血吸虫病肝纤维化的作用。
Background
Schistosomiasis japonicum is a severe endemic disease mainly prevalent in Central South China; it remains to be a serious zoonosis and seriously endanger people's health and effect social and economic development of epidemic areas. In the case of Schistosoma japonicum (S. japonicum) infection, the chronic onset of disease is not due to the adult worms but is chiefly related to the T-cell-dependent immune response of the host, which is directed against schistosome eggs trapped in tissues, mainly in the liver and intestines. The trapped eggs induce delayed hypersensitive reactions, evoking the formation of inflammatory granuloma and subsequent fibrosis. Liver fibrosis will further develop to cirrhosis, which causes death by supervene upper gastrointestinal hemorrhage or hepatic coma. Therefore, it is very neccesory to regular eggs-induced granuloma formation to prevent occurrence of advanced schistosomiasis japonica.
At present, praziquantel still is the only way to treat schistosomiasis in clinic. It could kill the schistosome, but not the eggs trapped in tissue. The eggs could cause granuloma formation by secreting antigen continually, and those schistosomiasis patients already have granuloma formation and subsequent fibrosis. The eggs-induced immune response will not terminate by treated with praziquantel. Interleukin-18(IL-18) is currently known cytokines for ameliorates treatment of schistosomiasis liver fibrosis. However, the approach of cytokines therapy need large amounts of particular cytokine, that will have serous side-effects and cause the immune system imbalance.
In our previous work, a S. japonicum single-chain fragment variable (SjscFv) specifically bound to the S.japonicum soluble immature egg antigen (SIEA) of26-28kDa was obtained from immunized mice, and its capability to target S.japonicum SIEA and soluble mature egg antigen (SEA) was confirmed. In present study, we will fuse this SjscFv with IL-18and clone into the eukaryotic vector pVAX1to produce SjscFv-IL18fusion protein, the specific S/scFv will targeting the IL-18to the site where parasitic eggs are embedded in the liver tissues and hepatic fibrosis is induced by SEA. Then the site-specific accumulation of functional IL-18will induce a predominant T-helper1(Thl) reaction, reduce the side-effects in normal tissue and induce more potent antifibrotic activity.
Objective
The objective of present study is to fuse SjscFv with IL-18and clone into the eukaryotic vector pVAX1and then transfect into macrophages. After treated with SEA, the supernatant of treated macrophages were used to incubate hepatic stellate cells (HSCs) and further observe the activation, proliferation and apoptosis of HSCs. The changes of extracellular matrix-related genes were used to assess its antifibrotic abilities in vitro. And use it as a therapeutic DNA vaccine to study its effect and mechanism on ameliorates hepatic fibrosis in vivo.
Methods
1. Identification of pVAX1/SjscFv-IL18plasmid
SjscFv and IL-18genes were linked using the splicing overlap extension PCR (SOE-PCR) method with the help of a glycine-rich linker consisting of15amino acids (Gly4Ser)3. The fusion gene SjscFv-IL18was cloned into the eukaryotic expression vector pVAXl which has been double digested with EcoR I and Xho I and transfected into E. coli BL21(DE3), and finally confirmed by double digestion with restriction enzymes and sequencing. Bioinformatics tools were used to predict its encoding protein structures.
2. pVAX1/SjscFv-IL18interfere the collagen generation and degradation in vitro
After recombinant eukaryotic plasmid transfected into macrophage, the supernate of SEA-treated macrophage were used to stimulate HSCs as conditioned media. The mRNA expression of those genes relatived with collagen generation and degradation in HSCs were analyzed by real time quantity PCR (qPCR). The proliferation and apoptotic of HSCs were measured by MTT and flow cytometric, respectively.
3. Effects of pVAX1/SjscFv-IL18on the mice liver fibrosis caused by schistosomiasis
Mice were challenged with S.japonicum cercariae infection on their abdominal skin. Forty-five days later, all infected mice were treated with praziquantel. At the same time, four kinds of plasmid were injected intramuscularly. Twenty wks after challenge, all mice were euthanized. The granuloma volume and collagen contents were estimated by H&E and Masson staining. IL-18and Thl/Th2cytokines level were analyzed by ELISA. TGF β1and IL-18in liver section were detected by immunohistochemistry (IHC). The mRNA expression of those genes relatived with collagen generation and degradation were analyzed by qPCR.
Results
1. Double digesting and sequencing confirmed the constructure of recombinant plasmid pVAX1/SjscFv-IL18. CD-search indicated that its encoding protein contained two Ig superfamily and one IL-1superfamily conserved domain.
2. pVAX1/SjscFv-IL18downregulated the mRNA expression of a-SMA, Col Ⅰ, Col Ⅲ, TIMP-1and upregulated the mRNA expression of MMPs-1in HSCs. It also could suppress proliferation and promote apoptotic in HSCs.
3. pVAX1/SjscFv-IL18could induce dominant Thl cytokines response in vivo. Consistent with the levels of Thl and Th2cytokines, mice vaccinated with pVAX1/SjscFv-IL18developed much less hepatic fibrosis, which was evaluated by average volumn of granuloma and collagen contents. It also could downregulate the mRNA expression of Col I, Col III, TIMP-1and upregulate the mRNA expression of MMPs-1in liver. Furthermore, pVAX1/SjscFv-IL18was more efficient than pVAX1/IL-18for its capability to deliver IL-18towards the site of hepatic fibrosis.
Conclusions
1. pVAX1/SjscFv-IL18was successfully constructed.
2. pVAX1/SjscFv-IL18can reduce collagen in vitro.
3. pVAX1/SjscFv-IL18can decrease hepatic fibrosis by delivering IL-18towards the site where the granuloma occur.
血吸虫病(schistosomiasis)是一种流行广泛、严重危害人类健康和社会经济发展的人兽共患寄生虫病,我国是日本血吸虫病(schistosomiasis japonica)流行最严重的四个国家之一。日本血吸虫(Schistosoma japonicum, Sj)致病的主要原因是雌虫产出的大量虫卵沉积于肝、肠等组织中,形成虫卵肉芽肿、组织损伤和继发性肝纤维化。肝纤维化的进一步发展导致肝硬化,患者可因上消化道出血、肝性昏迷等严重并发症而致死。因此,阻止虫卵肉芽肿病变的形成,对于控制日本血吸虫病的进程、防止晚期血吸虫病的发生具有重要的意义。
目前,临床上采用单一的吡喹酮治疗血吸虫病仍存在一定缺陷,吡喹酮只能杀灭童虫和成虫,而不能阻止肝组织内的虫卵继续分泌抗原,虫卵可溶性抗原(soluble matured egg antigen, SEA)可引起组织内虫卵肉芽肿反应,由于血吸虫病患者发现时多己转为慢性,肝组织内虫卵已经或正在形成虫卵肉芽肿或肝纤维化,有效杀虫治疗后病人依然有转化为晚期的可能,SEA诱导产生的免疫病理反应并不因杀虫治疗而终止。IL-18是目前己知的对血吸虫病肝纤维化具有治疗作用的细胞因子,然而单纯使用细胞因子治疗需连续、大剂量的用药,势必导致毒副作用的增加以及宿主体内细胞因子的严重失衡。
因此,我们拟利用实验室前期筛选得到的对日本血吸虫雌虫、未成熟虫卵、成熟虫卵等多个阶段均具有高特异性免疫靶向作用及识别能力的单链抗体(Sj single-chain Fv antibody, SjscFv),将其与对血吸虫性肝纤维化具有治疗作用的细胞因子IL-18融合表达,产生SjscFv-IL18融合蛋白,借助SjscFv的免疫及靶向作用携带IL-18进入虫卵肉芽肿和肝纤维化形成的局部,特异性提高局部免疫作用及IL-18浓度,诱导日本血吸虫感染慢性期从Th2型免疫应答转化为Th1型优势的免疫应答,同时能够极大地降低IL-18对其它正常组织器官的毒副作用,以期达到更好的抗日本血吸虫性病纤维化的效果。
研究目的
构建真核重组质粒pVAX1/SjscFv-IL18,转染巨噬细胞后,使用SEA刺激后的巨噬细胞上清液处理肝星状细胞(HSCs),观察对HSCs激活、增殖和凋亡的影响,并检测HSCs中与细胞外基质(ECM)沉积相关的基因表达,评估其抗肝纤维化的能力；并在日本血吸虫病肝纤维化小鼠中研究其对虫卵肉芽肿及肝纤维化形成的影响及其作用机制。
研究方法
1、真核重组质粒pVAX1/SjscFv-IL18的构建；
采用SOE-PCR技术构建重组质粒pVAX1/SjscFv-IL18和pVAX1/S/scFv,转化大肠杆菌后,进行大量表达,并通过双酶切和测序鉴定,使用生物信息学方法分析其结构特征。
2、pVAX1/SjscFv-IL18对HSCs胶原生成与降解的影响
将各组重组真核质粒转染巨噬细胞后,采用SEA刺激巨噬细胞后的上清液处理HSCs,应用实时荧光定量PCR(qPCR)检测胶原相关基因mRNA水平、MTT检测HSCs细胞增殖、流式细胞术检测HSCs凋亡。
3、pVAX1/SjscFv-IL18对日本血吸虫病肝纤维化的影响
建立日本血吸虫病肝纤维化小鼠模型,将pVAX1/SjscFv-IL18注入小鼠体内,采用]HE、Masson染色方法分别检测肝组织内虫卵肉芽肿的大小、肝组织内胶原沉积的情况,应用ELISA检测小鼠Thl/Th2型细胞因子、IL-18,免疫组化检测肝组织中TGF β1和IL-18, qPCR检测肝脏中胶原相关基因mRNA水平。
研究结果
1、PCR、双酶切及测序结果显示重组质粒插入序列与目的序列一致,CD-Search证实所构建质粒表达蛋白中包含目的蛋白所需的两个Ig超家族和IL-1超家族保守结构域,说明真核重组质粒pVAX1/SjscFv-IL18构建成功。
2. pVAX1/SjscFv-IL18转染巨噬细胞后,采用SEA刺激该巨噬细胞产生的上清液处理HSCs,与对照组相比,能够部分降低HSCs中a-SMA的mRNA水平,并抑制HSCs增殖、促进HSCs凋亡,上调MMPs-1的同时下调Col Ⅰ、Col Ⅲ和TIMP-1的mRNA表达。
3、pVAX1/SjscFv-IL18注入日本血吸虫病肝纤维化小鼠后,能够降低肝组织内虫卵肉芽肿的大小,减少肝组织内胶原的沉积,并在体内表达大量的IL18,诱导Thl型优势的免疫反应,减少肝脏中TGF β1表达,上调MMPs-1的同时下调Col Ⅰ、Col Ⅲ和TIMP-1的mRNA表达,且能够在虫卵肉芽肿局部提高IL-18的浓度,从而产生更好的抗日本血吸虫病肝纤维化的作用。
结论
1、成功构建了真核重组质粒pVAX1/SjscFv-IL18。
2、pVAX1/SjscFv-IL18在体外能够通过影响巨噬细胞内细胞因子的分泌进而抑制HSCs中胶原的生成,促进胶原降解。
3、pVAX1/SjscFv-IL18能够通过SjscFv的免疫靶向作用提高IL-18的生物学活性,从而抑制虫卵肉芽肿的形成,并产生更好的抗日本血吸虫病肝纤维化的作用。
Background
Schistosomiasis japonicum is a severe endemic disease mainly prevalent in Central South China; it remains to be a serious zoonosis and seriously endanger people's health and effect social and economic development of epidemic areas. In the case of Schistosoma japonicum (S. japonicum) infection, the chronic onset of disease is not due to the adult worms but is chiefly related to the T-cell-dependent immune response of the host, which is directed against schistosome eggs trapped in tissues, mainly in the liver and intestines. The trapped eggs induce delayed hypersensitive reactions, evoking the formation of inflammatory granuloma and subsequent fibrosis. Liver fibrosis will further develop to cirrhosis, which causes death by supervene upper gastrointestinal hemorrhage or hepatic coma. Therefore, it is very neccesory to regular eggs-induced granuloma formation to prevent occurrence of advanced schistosomiasis japonica.
At present, praziquantel still is the only way to treat schistosomiasis in clinic. It could kill the schistosome, but not the eggs trapped in tissue. The eggs could cause granuloma formation by secreting antigen continually, and those schistosomiasis patients already have granuloma formation and subsequent fibrosis. The eggs-induced immune response will not terminate by treated with praziquantel. Interleukin-18(IL-18) is currently known cytokines for ameliorates treatment of schistosomiasis liver fibrosis. However, the approach of cytokines therapy need large amounts of particular cytokine, that will have serous side-effects and cause the immune system imbalance.
In our previous work, a S. japonicum single-chain fragment variable (SjscFv) specifically bound to the S.japonicum soluble immature egg antigen (SIEA) of26-28kDa was obtained from immunized mice, and its capability to target S.japonicum SIEA and soluble mature egg antigen (SEA) was confirmed. In present study, we will fuse this SjscFv with IL-18and clone into the eukaryotic vector pVAX1to produce SjscFv-IL18fusion protein, the specific S/scFv will targeting the IL-18to the site where parasitic eggs are embedded in the liver tissues and hepatic fibrosis is induced by SEA. Then the site-specific accumulation of functional IL-18will induce a predominant T-helper1(Thl) reaction, reduce the side-effects in normal tissue and induce more potent antifibrotic activity.
Objective
The objective of present study is to fuse SjscFv with IL-18and clone into the eukaryotic vector pVAX1and then transfect into macrophages. After treated with SEA, the supernatant of treated macrophages were used to incubate hepatic stellate cells (HSCs) and further observe the activation, proliferation and apoptosis of HSCs. The changes of extracellular matrix-related genes were used to assess its antifibrotic abilities in vitro. And use it as a therapeutic DNA vaccine to study its effect and mechanism on ameliorates hepatic fibrosis in vivo.
Methods
1. Identification of pVAX1/SjscFv-IL18plasmid
SjscFv and IL-18genes were linked using the splicing overlap extension PCR (SOE-PCR) method with the help of a glycine-rich linker consisting of15amino acids (Gly4Ser)3. The fusion gene SjscFv-IL18was cloned into the eukaryotic expression vector pVAXl which has been double digested with EcoR I and Xho I and transfected into E. coli BL21(DE3), and finally confirmed by double digestion with restriction enzymes and sequencing. Bioinformatics tools were used to predict its encoding protein structures.
2. pVAX1/SjscFv-IL18interfere the collagen generation and degradation in vitro
After recombinant eukaryotic plasmid transfected into macrophage, the supernate of SEA-treated macrophage were used to stimulate HSCs as conditioned media. The mRNA expression of those genes relatived with collagen generation and degradation in HSCs were analyzed by real time quantity PCR (qPCR). The proliferation and apoptotic of HSCs were measured by MTT and flow cytometric, respectively.
3. Effects of pVAX1/SjscFv-IL18on the mice liver fibrosis caused by schistosomiasis
Mice were challenged with S.japonicum cercariae infection on their abdominal skin. Forty-five days later, all infected mice were treated with praziquantel. At the same time, four kinds of plasmid were injected intramuscularly. Twenty wks after challenge, all mice were euthanized. The granuloma volume and collagen contents were estimated by H&E and Masson staining. IL-18and Thl/Th2cytokines level were analyzed by ELISA. TGF β1and IL-18in liver section were detected by immunohistochemistry (IHC). The mRNA expression of those genes relatived with collagen generation and degradation were analyzed by qPCR.
Results
1. Double digesting and sequencing confirmed the constructure of recombinant plasmid pVAX1/SjscFv-IL18. CD-search indicated that its encoding protein contained two Ig superfamily and one IL-1superfamily conserved domain.
2. pVAX1/SjscFv-IL18downregulated the mRNA expression of a-SMA, Col Ⅰ, Col Ⅲ, TIMP-1and upregulated the mRNA expression of MMPs-1in HSCs. It also could suppress proliferation and promote apoptotic in HSCs.
3. pVAX1/SjscFv-IL18could induce dominant Thl cytokines response in vivo. Consistent with the levels of Thl and Th2cytokines, mice vaccinated with pVAX1/SjscFv-IL18developed much less hepatic fibrosis, which was evaluated by average volumn of granuloma and collagen contents. It also could downregulate the mRNA expression of Col I, Col III, TIMP-1and upregulate the mRNA expression of MMPs-1in liver. Furthermore, pVAX1/SjscFv-IL18was more efficient than pVAX1/IL-18for its capability to deliver IL-18towards the site of hepatic fibrosis.
Conclusions
1. pVAX1/SjscFv-IL18was successfully constructed.
2. pVAX1/SjscFv-IL18can reduce collagen in vitro.
3. pVAX1/SjscFv-IL18can decrease hepatic fibrosis by delivering IL-18towards the site where the granuloma occur.
引文
[1]Steinmann P, Keiser J, Bos R, et al. Schistosomiasis and water resources development:systematic review, meta-analysis, and estimates of people at risk[J]. The Lancet Infectious Diseases,2006,6(7):411-425.
[2]Chitsulo L, Engels D, Montresor A, et al. The global status of schistosomiasis and its control[J]. Acta Trop,2000,77(1):41-51.
[3]McManus DP, Loukas A. Current status of vaccines for schistosomiasis[J]. Clin Microbiol Rev,2008,21(1):225-242.
[4]Prevention and control of schistosomiasis and soil-transmitted helminthiasis[J]. World Health Organ Tech Rep Ser,2002,912:i-vi,1-57, back cover.
[5]King CH, Dickman K, Tisch DJ. Reassessment of the cost of chronic helmintic infection:a meta-analysis of disability-related outcomes in endemic schistosomiasis[J]. Lancet,2005,365(9470):1561-1569.
[6]Zhou XN, Guo JG, Wu XH, et al. Epidemiology of schistosomiasis in the People's Republic of China,2004[J]. Emerg Infect Dis,2007,13(10): 1470-1476.
[7]郝阳,郑洁,朱蓉,等.2009年全国血吸虫病疫情通报[J].中国血吸虫病防治杂志,2010,22：521-527.
[8]汪世平主编.医学寄生虫学[M].北京：高等教育出版社,2009.
[9]Giboda M, Smith JM. Schistosoma mansoni eggs as a target for praziquantel: efficacy of oral application in mice[J]. J Trop Med Hyg,1994,97(2):98-102.
[10]Matsuda H, Tanaka H, Nogami S, et al. Mechanism of action of praziquantel on the eggs of Schistosoma japonicum[J]. Jpn J Exp Med,1983,53(6): 271-274.
[11]陈峰,蔡为民,陈智,等.血吸虫病肝纤维的患者转化生长因子β1mRNA的水平极其临床意义[J].中国寄生虫学与寄生虫病杂志,1999,17(3)：140.
[12]蔡卫民,张立煌,孙永良,等.日本血吸虫病肝纤维化患者免疫调节有关因素的探讨[J].中华传染病杂志,1993,11：63.
[13]Rockey DC. Hepatic fibrosis, stellate cells, and portal hypertension[J]. Clin Liver Dis,2006,10(3):459-479, vii-viii.
[14]Ramachandran P, Iredale JP. Reversibility of liver fibrosis[J]. Ann Hepatol, 2009,8(4):283-291.
[15]Chiang DJ, Pritchard MT, Nagy LE. Obesity, diabetes mellitus, and liver fibrosis[J]. Am J Physiol Gastrointest Liver Physiol,2011,300(5):G697-702.
[16]Bartley PB, Ramm GA, Jones MK, et al. A contributory role for activated hepatic stellate cells in the dynamics of Schistosoma japonicum egg-induced fibrosis[J]. Int J Parasitol,2006,36(9):993-1001.
[17]Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis[J]. J Gastroenterol Hepatol,2007,22 Suppl 1:S79-84.
[18]Chu D, Li C, Wu Q, et al. Paeoniflorin prevents hepatic fibrosis of Schistosomiasis japonica by inhibiting TGF-β1 production from macrophages in mice[J]. Frontiers of Medicine in China,2008,2(2):154-165.
[19]Yang J, Zheng J, Wu L, et al. NDRG2 ameliorates hepatic fibrosis by inhibiting the TGF-betal/Smad pathway and altering the MMP2/TIMP2 ratio in rats[J]. PLoS One,2011,6(11):e27710.
[20]Yoshida K, Matsuzaki K. Differential Regulation of TGF-beta/Smad Signaling in Hepatic Stellate Cells between Acute and Chronic Liver Injuries[J]. Front Physiol,2012,3:53.
[21]Ahmad ZA, Yeap SK, Ali AM, et al. scFv Antibody:Principles and Clinical Application[J]. Clin Dev Immunol,2012,2012:980250.
[22]Tang LX, He RH, Yang G, et al. Asiatic acid inhibits liver fibrosis by blocking TGF-beta/Smad signaling in vivo and in vitro[J]. PLoS One,2012,7(2): e31350.
[23]Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling[J]. Nature,2003,425(6958):577-584.
[24]Pearce EJ, Caspar P, Grzych JM, et al. Downregulation of Thl cytokine production accompanies induction of Th2 responses by a parasitic helminth, Schistosoma mansoni[J]. J Exp Med,1991,173(1):159-166.
[25]Grzych JM, Pearce E, Cheever A, et al. Egg deposition is the major stimulus for the production of Th2 cytokines in murine schistosomiasis mansoni[J]. J Immunol,1991,146(4):1322-1327.
[26]Vella AT, Pearce EJ. CD4+ Th2 response induced by Schistosoma mansoni eggs develops rapidly, through an early, transient, ThO-like stage[J]. J Immunol,1992,148(7):2283-2290.
[27]Kent G, Gay S, Inouye T, et al. Vitamin A-containing lipocytes and formation of type III collagen in liver injury[J]. Proc Natl Acad Sci U S A,1976,73(10): 3719-3722.
[28]Maher JJ, McGuire RF. Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo[J]. J Clin Invest,1990,86(5):1641-1648.
[29]Milani S, Herbst H, Schuppan D, et al. Cellular localization of type Ⅰ Ⅲ and IV procollagen gene transcripts in normal and fibrotic human liver[J]. Am J Pathol,1990,137(1):59-70.
[30]Casini A, Pinzani M, Milani S, et al. Regulation of extracellular matrix synthesis by transforming growth factor beta 1 in human fat-storing cells [J]. Gastroenterology,1993,105(1):245-253.
[31]Chiaramonte MG, Cheever AW, Malley JD, et al. Studies of murine schistosomiasis reveal interleukin-13 blockade as a treatment for established and progressive liver fibrosis[J]. Hepatology,2001,34(2):273-282.
[32]Gressner AM, Weiskirchen R, Breitkopf K, et al. Roles of TGF-beta in hepatic fibrosis[J]. Front Biosci,2002,7:d793-807.
[33]De Bleser PJ, Jannes P, van Buul-Offers SC, et al. Insulinlike growth factor-II/mannose 6-phosphate receptor is expressed on CC14-exposed rat fat-storing cells and facilitates activation of latent transforming growth factor-beta in cocultures with sinusoidal endothelial cells[J]. Hepatology,1995, 21(5):1429-1437.
[34]Horras CJ, Lamb CL, Mitchell KA. Regulation of hepatocyte fate by interferon-gamma[J]. Cytokine Growth Factor Rev,2011,22(1):35-43.
[35]Tao FF, Yang YF, Wang H, et al. Thl-type epitopes-based cocktail PDDV attenuates hepatic fibrosis in C57BL/6 mice with chronic Schistosoma japonicum infection[J]. Vaccine,2009,27(31):4110-4117.
[36]Dooley S, Said HM, Gressner AM, et al. Y-box protein-1 is the crucial mediator of antifibrotic interferon-gamma effects[J]. J Biol Chem,2006, 281(3):1784-1795.
[37]Weng H, Mertens PR, Gressner AM, et al. IFN-gamma abrogates profibrogenic TGF-beta signaling in liver by targeting expression of inhibitory and receptor Smads[J]. J Hepatol,2007,46(2):295-303.
[38]Weng HL, Ciuclan L, Liu Y, et al. Profibrogenic transforming growth factor-beta/activin receptor-like kinase 5 signaling via connective tissue growth factor expression in hepatocytes[J]. Hepatology,2007,46(4): 1257-1270.
[39]Zhang L, Mi J, Yu Y, et al. IFN-gamma gene therapy by intrasplenic hepatocyte transplantation:a novel strategy for reversing hepatic fibrosis in Schistosoma japonicum-infectsd mice[J]. Parasite Immunol,2001,23(1): 11-17.
[40]Ushio S, Namba M, Okura T, et al. Cloning of the cDNA for human IFN-gamma-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein[J]. J Immunol,1996,156(11):4274-4279.
[41]Wei F, Liu Q, Gao S, et al. Enhancement by IL-18 of the protective effect of a Schistosoma japonicum 26kDa GST plasmid DNA vaccine in mice[J]. Vaccine, 2008,26(33):4145-4149.
[42]Wei F, Liu Q, Zhai Y, et al. IL-18 enhances protective effect in mice immunized with a Schistosoma japonicum FABP DNA vaccine[J]. Acta Trop, 2009,111(3):284-288.
[43]Zhang LH, Pan JP, Yao HP, et al. Intrasplenic transplantation of IL-18 gene-modified hepatocytes:an effective approach to reverse hepatic fibrosis in schistosomiasis through induction of dominant Thl response[J]. Gene Ther, 2001,8(17):1333-1342.
[44]Rockey DC, Maher JJ, Jarnagin WR, et al. Inhibition of rat hepatic lipocyte activation in culture by interferon-gamma[J]. Hepatology,1992,16(3): 776-784.
[45]Tiggelman AM, Boers W, Linthorst C, et al. Collagen synthesis by human liver (myo)fibroblasts in culture:evidence for a regulatory role of IL-1 beta, IL-4, TGF beta and IFN gamma[J]. J Hepatol,1995,23(3):307-317.
[46]Baroni GS, D'Ambrosio L, Curto P, et al. Interferon gamma decreases hepatic stellate cell activation and extracellular matrix deposition in rat liver fibrosis[J]. Hepatology,1996,23(5):1189-1199.
[47]Rockey DC, Chung JJ. Interferon gamma inhibits lipocyte activation and extracellular matrix mRNA expression during experimental liver injury: implications for treatment of hepatic fibrosis[J]. J Investig Med,1994,42(4): 660-670.
[48]Toyonaga T, Hino O, Sugai S, et al. Chronic active hepatitis in transgenic mice expressing interferon-gamma in the liver[J]. Proc Natl Acad Sci U S A,1994, 91(2):614-618.
[49]Weisser NE, Hall JC. Applications of single-chain variable fragment antibodies in therapeutics and diagnostics[J]. Biotechnol Adv,2009,27(4): 502-520.
[50]Lee YC, Leu SJ, Hung HC, et al. A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage[J]. Vet Immunol Immunopathol,2007,117(1-2): 75-85.
[51]Das D, Kriangkum J, Nagata LP, et al. Development of a biotin mimic tagged ScFv antibody against western equine encephalitis virus:bacterial expression and refolding[J]. J Virol Methods,2004,117(2):169-177.
[52]Ascione A, Capecchi B, Campitelli L, et al. Human monoclonal antibodies in single chain fragment variable format with potent neutralization activity against influenza virus H5N1[J]. Antiviral Res,2009,83(3):238-244.
[53]Hu S, Li L, Qiao J, et al. Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris[J]. Protein Expr Purif,2006,47(1):249-257.
[54]Walper SA, Anderson GP, Brozozog Lee PA, et al. Rugged Single Domain Antibody Detection Elements for Bacillus anthracis Spores and Vegetative Cells[J]. PLoS One,2012,7(3):e32801.
[55]汪世平,赵慰先,易新元,等.日本血吸虫未成熟虫卵可溶性抗原诱导抗卵 免疫初步研究[J].湖南医科大学学报,1995,20(3)：193-196.
[56]汪世平,赵慰先,周泪波,等.日本血吸虫卵的体外培养与抗卵胚发育研究[J].中华医学杂志,1996,76(3)：218-221.
[57]汪世平,赵慰先,易新元,等.日本血吸虫未成熟卵可溶性抗原的初步分析[J].中国人兽共患病杂志,1995,11(4)：35-37.
[58]汪世平,周泪波,张顺科,等.日本血吸虫未成熟虫卵对小鼠肝肉芽肿形成的免疫学影响[J].中华医学杂志,1997,77(10)：768-770.
[59]汪世平,周泪波,沈国励,等.日本血吸虫未成熟虫卵26/28kDa抗原诱导抗雌虫生殖和抗卵胚发育免疫的研究[J].中国寄生虫学与寄生虫病杂志,1997,15(2)：79-83.
[60]汪世平,周泪波,曾宪芳,等.抗日本血吸虫生殖及卵胚发育免疫的研究[J].中国血吸虫病防治杂志,1998,10：7-12.
[61]汪世平,周泪波,曾宪芳,等.抗日本血吸虫未成熟虫卵部分纯化抗原免疫血清的制备及定位观察[J].中华传染病杂志,1997,15(3)：155-156.
[62]He Z, Wang SP, Xiao XQ, et al. Construction screening and application of single-chain Fv antibody library against immature egg of Schistosoma japonicum[J]. Prog Biochem Biophys,2008,35:921-928.
[63]Kozak M. The scanning model for translation:an update[J]. J Cell Biol,1989, 108(2):229-241.
[64]Stewart CA, Horton R, Allcock RJ, et al. Complete MHC haplotype sequencing for common disease gene mapping[J]. Genome Res,2004,14(6): 1176-1187.
[65]萨姆布克J,拉塞尔WD,黄培堂,等.分子克隆实验指南.第三版[M].北京：科学出版社,2002,8.
[66]Bradbury AR, Marks JD. Antibodies from phage antibody libraries[J]. J Immunol Methods,2004,290(1-2):29-49.
[67]Marasco WA, Haseltine WA, Chen SY. Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody [J]. Proc Natl Acad Sci U S A,1993,90(16):7889-7893.
[68]Biocca S, Pierandrei-Amaldi P, Campioni N, et al. Intracellular immunization with cytosolic recombinant antibodies[J]. Biotechnology (N Y),1994,12(4): 396-399.
[69]Cohen PA. Intrabodies. Targeting scFv expression to eukaryotic intracellular compartments[J]. Methods Mol Biol,2002,178:367-378.
[70]Gao DM, Wang SP, He Z, et al. Schistosoma japonicum:screening of cercariae cDNA library by specific single-chain antibody against SIEA26-28 ku and immunization experiment of the recombinant plasmids containing the selected genes[J]. Parasitol Res,2010,107(1):127-134.
[71]高冬梅.SIEA26-28ku-scFv抗日本血吸虫病治疗性疫苗的免疫靶向作用及其效果的初步观察[D].长沙：中南大学,2010.
[72]Heuser C, Guhlke S, Matthies A, et al. Anti-CD30-scFv-Fc-IL-2 antibody-cytokine fusion protein that induces resting NK cells to highly efficient cytolysis of Hodgkin's lymphoma derived tumour cells[J]. Int J Cancer,2004,110(3):386-394.
[73]Halin C, Gafner V, Villani ME, et al. Synergistic therapeutic effects of a tumor targeting antibody fragment, fused to interleukin 12 and to tumor necrosis factor alpha[J]. Cancer Res,2003,63(12):3202-3210.
[74]Helguera G, Dela Cruz JS, Lowe C, et al. Vaccination with novel combinations of anti-HER2/neu cytokines fusion proteins and soluble protein antigen elicits a protective immune response against HER2/neu expressing tumors[J]. Vaccine,2006,24(3):304-316.
[75]Higuchi R, Krummel B, Saiki RK. A general method of in vitro preparation and specific mutagenesis of DNA fragments:study of protein and DNA interactions[J]. Nucleic Acids Res,1988,16(15):7351-7367.
[76]Lu Q. Seamless cloning and gene fusion[J]. Trends Biotechnol,2005,23(4): 199-207.
[77]Gustavsson M, Lehtio J, Denman S, et al. Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris[J]. Protein Eng,2001,14(9):711-715.
[78]Huston JS, Tai MS, McCartney J, et al. Antigen recognition and targeted delivery by the single-chain Fv[J]. Cell Biophys,1993,22(1-3):189-224.
[79]Arnold K, Bordoli L, Kopp J, et al. The SWISS-MODEL workspace:a web-based environment for protein structure homo logy modelling[J]. Bioinformatics,2006,22(2):195-201.
[80]Schwede T, Kopp J, Guex N, et al. SWISS-MODEL:An automated protein homology-modeling server[J]. Nucleic Acids Res,2003,31(13):3381-3385.
[81]Schwede T, Diemand A, Guex N, et al. Protein structure computing in the genomic era[J]. Res Microbiol,2000,151(2):107-112.
[82]Bazan JF, Timans JC, Kastelein RA. A newly defined interleukin-1?[J]. Nature, 1996,379(6566):591.
[83]Marchler-Bauer A, Lu S, Anderson JB, et al. CDD:a Conserved Domain Database for the functional annotation of proteins [J]. Nucleic Acids Res,2011, 39(Database issue):D225-229.
[84]Marchler-Bauer A, Bryant SH. CD-Search:protein domain annotations on the fly[J]. Nucleic Acids Res,2004,32(Web Server issue):W327-331.
[85]Marchler-Bauer A, Anderson JB, Chitsaz F, et al. CDD:specific functional annotation with the Conserved Domain Database[J]. Nucleic Acids Res,2009, 37(Database issue):D205-210.
[86]Okamura H, Tsutsi H, Komatsu T, et al. Cloning of a new cytokine that induces IFN-gamma production by T cells[J]. Nature,1995,378(6552):88-91.
[87]Wigler M, Silverstein S, Lee LS, et al. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells[J]. Cell,1977,11(1):223-232.
[88]Sun CJ, Pan SP, Xie QX, et al. Preparation of chitosan-plasmid DNA nanoparticles encoding zona pellucida glycoprotein-3alpha and its expression in mouse[J]. Mol Reprod Dev,2004,68(2):182-188.
[89]Huntley JF, Stabel JR, Paustian ML, et al. Expression library immunization confers protection against Mycobacterium avium subsp. paratuberculosis infection[J]. Infect Immun,2005,73(10):6877-6884.
[90]Duran EM, Shapshak P, Worley J, et al. Presenilin-1 detection in brain neurons and FOXP3 in peripheral blood mononuclear cells:normalizer gene selection for real time reverse transcriptase pcr using the deltadeltaCt method[J]. Front Biosci,2005,10:2955-2965.
[91]Matsuoka M, Tsukamoto H. Stimulation of hepatic lipocyte collagen production by Kupffer cell-derived transforming growth factor beta: implication for a pathogenetic role in alcoholic liver fibrogenesis[J]. Hepatology,1990,11(4):599-605.
[92]Bachem MG, Meyer D, Melchior R, et al. Activation of rat liver perisinusoidal lipocytes by transforming growth factors derived from myofibroblastlike cells. A potential mechanism of self perpetuation in liver fibrogenesis[J]. J Clin Invest,1992,89(1):19-27.
[93]Bissell DM, Wang SS, Jarnagin WR, et al. Cell-specific expression of transforming growth factor-beta in rat liver. Evidence for autocrine regulation of hepatocyte proliferation[J]. J Clin Invest,1995,96(1):447-455.
[94]Bachem MG, Riess U, Melchior R, et al. Transforming growth factors (TGF alpha and TGF beta 1) stimulate chondroitin sulfate and hyaluronate synthesis in cultured rat liver fat storing cells[J]. FEBS Lett,1989,257(1):134-137.
[95]Qi Z, Atsuchi N, Ooshima A, et al. Blockade of type beta transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat[J]. Proc Natl Acad Sci U S A,1999,96(5):2345-2349.
[96]George J, Roulot D, Koteliansky VE, et al. In vivo inhibition of rat stellate cell activation by soluble transforming growth factor beta type II receptor:a potential new therapy for hepatic fibrosis[J]. Proc Natl Acad Sci U S A,1999, 96(22):12719-12724.
[97]Yata Y, Gotwals P, Koteliansky V, et al. Dose-dependent inhibition of hepatic fibrosis in mice by a TGF-beta soluble receptor:implications for antifibrotic therapy[J]. Hepatology,2002,35(5):1022-1030.
[98]Zhang BB, Jiao YW, Cai WM, et al. Influence of interferon gamma treatment on expression of TGF-betal and its receptors in liver fibrosis of mice with schistosomiasis japonica[J]. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi,2004,22(6):340-343.
[99]Paiva LA, Maya-Monteiro CM, Bandeira-Melo C, et al. Interplay of cysteinyl leukotrienes and TGF-beta in the activation of hepatic stellate cells from Schistosoma mansoni granulomas[J]. Biochim Biophys Acta,2010,1801(12): 1341-1348.
[100]Chu D, Luo Q, Li C, et al. Paeoniflorin inhibits TGF-beta1-mediated collagen production by Schistosoma japonicum soluble egg antigen in vitro [J]. Parasitology,2007,134(Pt 11):1611-1621.
[101]Li J, Wang W, Shen JL. The role of TGFbetal and IL-13 in cellular signal transduction of hepatic fibrosis of schistosomiasis[J]. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi,2009,27(4):357-360.
[102]Zhang JP, Zhang M, Jin C, et al. Matrine inhibits production and actions of fibrogenic cytokines released by mouse peritoneal macrophages[J]. Acta Pharmacol Sin,2001,22(8):765-768.
[103]Friedman SL. Hepatic Fibrosis. In:Schiff ER, Sorrell MF, Maddrey WC, eds. Schiff's Diseases of the Liver[J]. Lippincott Williams & Wilkins,2003: 409-427.
[104]Eng FJ, Friedman SL. Fibrogenesis I. New insights into hepatic stellate cell activation:the simple becomes complex[J]. Am J Physiol Gastrointest Liver Physiol,2000,279(1):G7-G11.
[105]Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury[J]. J Biol Chem,2000,275(4):2247-2250.
[106]Darwich L, Coma G, Pena R, et al. Secretion of interferon-gamma by human macrophages demonstrated at the single-cell level after costimulation with interleukin (IL)-12 plus IL-18[J]. Immunology,2009,126(3):386-393.
[107]Bansal R, Prakash J, de Ruijter M, et al. Peptide-modified albumin carrier explored as a novel strategy for a cell-specific delivery of interferon gamma to treat liver fibrosis[J]. Mol Pharm,2011,8(5):1899-1909.
[108]Ha MH, Wei L, Rao HY, et al Effect of interferon-gamma on hepatic stellate cells stimulated by acetaldehyde[J]. Hepatogastroenterology,2008,55(84): 1059-1065.
[109]Schmitt-Graff A, Kruger S, Bochard F, et al. Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers[J]. Am J Pathol,1991,138(5):1233-1242.
[110]Carpino G, Morini S, Ginanni Corradini S, et al Alpha-SMA expression in hepatic stellate cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis after liver transplantation[J]. Dig Liver Dis, 2005,37(5):349-356.
[111]Iwaisako K, Brenner DA, Kisseleva T. What's new in liver fibrosis? The origin of myofibroblasts in liver fibrosis[J]. J Gastroenterol Hepatol,2012,27 Suppl 2:65-68.
[112]Qu Y, Chen WH, Zong L, et al.18alpha-Glycyrrhizin induces apopto sis and suppresses activation of rat hepatic stellate cells [J]. Med Sci Monit,2012, 18(1):BR24-32.
[113]Ping J, Gao AM, Qin HQ, et al. Indole-3-carbinol enhances the resolution of rat liver fibrosis and stimulates hepatic stellate cell apopto sis by blocking the inhibitor of kappaB kinase alpha/inhibitor of kappaB-alpha/nuclear factor-kappaB pathway[J]. J Pharmacol Exp Ther,2011,339(2):694-703.
[114]Friedman SL, Roll FJ, Boyles J, et al Hepatic lipocytes:the principal collagen-producing cells of normal rat liver[J]. Proc Natl Acad Sci U S A, 1985,82(24):8681-8685.
[115]Bedossa P, Paradis V. Approaches for treatment of liver fibrosis in chronic hepatitis C[J]. Clin Liver Dis,2003,7(1):195-210.
[116]Roderfeld M, Hemmann S, Roeb E. Mechanisms of fibrinolysis in chronic liver injury (with special emphasis on MMPs and TIMPs)[J]. Z Gastroenterol, 2007,45(1):25-33.
[117]Murphy FR, Issa R, Zhou X, et al. Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of metalloproteinase-1 is mediated via effects on matrix metalloproteinase inhibition:implications for reversibility of liver fibrosis[J]. J Biol Chem,2002,277(13):11069-11076.
[118]Fowell AJ, Collins JE, Duncombe DR, et al Silencing tissue inhibitors of metalloproteinases (TIMPs) with short interfering RNA reveals a role for TIMP-1 in hepatic stellate cell proliferation[J]. Biochem Biophys Res Commun,2011,407(2):277-282.
[119]Fabre V, Wu H, PondTor S, et al. Tissue inhibitor of matrix-metalloprotease-1 predicts risk of hepatic fibrosis in human Schistosoma japonicum infection[J]. J Infect Dis,2011,203(5):707-714.
[120]Tian Z, Wang SP, Zeng SH, et al. Characterization and vaccination of two novel Schistosoma japonicum genes screened from a cercaria cDNA library[J]. Parasitol Res,2012,110(1):403-409.
[121]Cheever AW, Duvall RH, Hallack TA, Jr., et al. Variation of hepatic fibrosis and granuloma size among mouse strains infected with Schistosoma mansoni[J]. Am J Trop Med Hyg,1987,37(1):85-97.
[122]Kofta W, Wedrychowicz H. c-DNA vaccination against parasitic infections: advantages and disadvantages[J]. Vet Parasitol,2001,100(1-2):3-12.
[123]Dinarello CA, Fantuzzi G. Interleukin-18 and host defense against infection[J]. J Infect Dis,2003,187 Suppl 2:S370-384.
[124]Hou X, Yu F, Man S, et al. Negative regulation of Schistosoma japonicum egg-induced liver fibrosis by natural killer cells [J]. PLoS Negl Trop Dis,2012, 6(1):e1456.
[125]Pearce EJ, MacDonald AS. The immunobiology of schistosomiasis[J]. Nat Rev Immunol,2002,2(7):499-511.
[126]Kim HJ, Song SB, Choi JM, et al. IL-18 downregulates collagen production in human dermal fibroblasts via the ERK pathway[J]. J Invest Dermatol,2010, 130(3):706-715.
[127]Arthur MJ. Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis[J]. Am J Physiol Gastrointest Liver Physiol,2000,279(2):G245-249.
[128]McCrudden R, Iredale JP. Liver fibrosis, the hepatic stellate cell and tissue inhibitors of metalloproteinases [J]. Histol Histopathol,2000,15(4): 1159-1168.
[129]Singh KP, Gerard HC, Hudson AP, et al. Expression of matrix metalloproteinases and their inhibitors during the resorption of schistosome egg-induced fibrosis in praziquantel-treated mice[J]. Immunology,2004, 111(3):343-352.
[130]Rhyner C, Konthur Z, Blaser K, et al. High-throughput isolation of recombinant antibodies against recombinant allergens[J]. Biotechniques,2003, 35(4):672-674.
[131]Menzel C, Schirrmann T, Konthur Z, et al. Human antibody RNase fusion protein targeting CD30+ lymphomas[J]. Blood,2008,111(7):3830-3837.
[132]Kim SH, Cho D, Kim TS. Anti-CD3 single-chain Fv/interleukin-18 fusion DNA induces anti-myco bacterial resistance via efficient interferon-gamma production in BALB/c mice infected with Mycobacterium avium[J]. Vaccine, 2006,24(16):3365-3373.
[133]Gillies SD, Young D, Lo KM, et al Biological activity and in vivo clearance of antitumor antibody/cytokine fusion proteins[J]. Bioconjug Chem,1993, 4(3):230-235.
[1]Matthias Peipp, Domenica Saul, Karin Barbin, et al. Efficient eukaryotic expression of fluorescent scFv fusion proteins directed against CD antigens for FACS applications [J]. Journal of Immunological Methods,2004, 285:265-280.
[2]Nina E. Weisser, J. Christopher Hall. Applications of single-chain variable fragment antibodies in therapeutics and diagnostics [J]. Biotechnology Advances,2009,27:502-520.
[3]KurtC. Almquist, Michael D. McLean, Yongqing Niu, et al. Expression of an anti-botulinum toxin A neutralizing single-chain Fv recombinant antibody in transgenic tobacco [J]. Vaccine,2006,24:2079-2086.
[4]Yu-Ching Lee, Sy-Jye C. Leu, Han-Chang Hung, et al. A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage [J]. Veterinary Immunology and Immunopathology,2007,117:75-85.
[5]D. Das, J. Kriangkum, L.P. Nagata, et al. Development of a biotin mimic tagged ScFv antibody against western equine encephalitis virus:bacterial expression and refolding [J]. Journal of Virological Methods,2004, 117:169-177.
[6]Alessandro Ascione, Barbara Capecchi, Laura Campitelli, et al. Human monoclonal antibodies in single chain fragment variable format with potent neutralization activity against influenza virus H5N1 [J]. Antiviral Research, 2009,2494:1-7.
[7]赵泽文,梁志清,史常旭等.抗rhEndoglin单链抗体的可溶性表达、纯化及活性鉴定[J].免疫学杂志,2009,25(1)：56-60.
[8]张桂红,刘彦仿,付勇等.抗肝癌单链抗体免疫脂质体的制备及其体外抑瘤实验[J].医学研究生学报,2006,19(1)：3-5.
[9]Siyi Hu, Liangwei Li, Jingjuan Qiao, et al. Codon optimization,expression,and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris [J]. Protein Expression and Purication,2006,47:249-257.
[10]祝怀平,王迎春,季顺东等.抗vWF与GPIb2IX结合部位的噬菌体呈现型单链抗体制备及功能研究[J].中国免疫学杂,2005,21：308-402.
[11]郭安,尹继刚,向梅等. 微小隐孢子虫子孢子与宿主黏附相关蛋白的筛 选[J].中国寄生虫学与寄生虫病杂志,2009,27(1)：87-88.
[12]L.-N. HOE, K.-L. WAN, S. NATHAN.Construction and characterization of recombinant a single-chain variable fragment antibodies against Toxoplasma gondii MIC2 protein [J]. Parasitology 2005,131:759-768.
[13]Jorge Sepulveda, Charles B. Shoemaker. Design and testing of PCR primers for the construction of scFv libraries representing the immunoglobulin repertoire of rats [J]. Journal of Immunological Methods,2008,332:92-102.
[14]Susana Re'fega, Marianne Cluzeaud, Pierre Pe'ry, et al. Production of a functional chicken single-chain variable fragment antibody derived from caecal tonsils B lymphocytes against macrogamonts of Eimeria tenella [J]. Veterinary Immunology and Immunopathology,2004,97:219-230
[15]司进,朱荫昌,曹利民等.抗弓形虫主要表面抗原1单链抗体的筛选及鉴定[J].中国血吸虫病防止杂志,2005,17(3)：202-206.
[16]陈新华,温浩,刘辉等.抗棘球蚴人源单链可变区抗体抑制人体包虫原头节的初步实验[J].中华微生物学和免疫学杂志,2004,24(9)：699-702.
[17]Heather Griffin, Robert Elston, Deborah Jackson, et al. Inhibition of Papillomavirus Protein Function in Cervica Cancer Cells by Intrabody Targeting [J]. J. Mol. Biol. 2006,355:360-378.
[18]Gregory P. White, Els N.T. Meeusen, Susan E. Newton. A single-chain variable region immunoglobulin library from the abomasal lymph node of sheep infected with the gastrointestinal nematode parasite Haemonchus contortus [J]. Veterinary Immunology and Immunopathology,2001, 78:117-129.
[19]Shigeto Yoshida, Hiroyuki Matsuoka, Enjie Luo, et al. A single-chain antibody fragment specific for the Plasmodium berghei ookinete protein Pbs21 confers transmission blockade in the mosquito midgut [J]. Molecular and Biochemical Parasitology,1999,104:195-204.
[20]So-Hee Kim, Seung-Young Hwang, Yong-Seok Lee, et al. Single-Chain Antibody Fragment Specific for Plasmodium vivax Duff Binding Protein [J]. CLINICAL AND VACCINE IMMUNOLOGY,2007,14(6):726-731.
[21]David R. Maass, Gavin B.L. Harrison, Warwick N. Grant, et al. Three surface antigens dominate the mucosal antibody response to gastrointestinal L3-stage strongylid nematodes in field immune sheep [J]. International Journal for Parasitology,2007,37:953-962.
[22]Guilherme Rocha Lino De Souza, Carlos Roberto Prudencio, Rone Cardoso, et al. Construction of an antibodies library(scFv) for selection and characterization of Boophilus microplus antigens [J]. Abstracts/Veterinary Immunology and Immunopathology,2009,128:211-347.
[23]何卓,汪世平,周帅锋等. 抗日本血吸虫生殖功能分子SIEA26-28kDa单链抗体与EGFP的融合表达及靶向性研究[J].中国人兽共患病学报,2008,24(8)：704-707.
[24]何卓,汪世平,肖小芹等.日本血吸虫未成熟卵单链抗体库的构建、筛选及初步应用[J].生物化学与生物物理进展,2008,35(8)：921-928.
[25]Petra Pavlickova, E. Marion Schneider, Hubert Hug. Advances in recombinant antibody microarrays [J]. Clinica Chimica Acta,2004,343:17-35.
[26]CHEN Dai-xiong, HE Ai, ZHAN Xi-mei, et al. Schistosoma japonicum: construction of phage display antibody library and its application in the immunodiagnosis of infection [J]. Chinese Medical Journal,2004, 117(11):1697-1703.
[27]尹继刚,张西臣,李建华等.抗隐孢子虫子孢子ScFv-PE40免疫毒素表达质粒的构建及其在大肠杆菌中的表达[J].寄生虫与医学昆虫学报,2005,12(3)：139-142.
[28]刘晓峰,李淑红,张西臣等.抗隐孢子虫ScFv-PE重组毒素原核表达载体的构建及其对虫体的杀灭作用[J].热带医学杂志,2007,7(4)：307-310.
[29]司进,朱荫昌,曹利民等.弓形虫主要表面抗原1单链抗体与绿色荧光蛋白的融合表达及其生物活性初步观察[J].中国寄生虫学与寄生虫病杂志,2006,24(3)：161-165.
[30]丁雪冰,陈晓光.恶性疟原虫环子孢子蛋白单链抗体与按蚊cecropin A融合基因的构建、原核表达及生物学活性的初步分析[J].寄生虫与医学昆虫学报,2008,15(1)：8-13.
[31]司进,朱荫昌,曹利民等.抗弓形虫靶向抗菌肽的构建及其效应的初步评价[J].中国血吸虫病防治杂志,2005,17(5)：356-361.
[32]Shigeto Yoshida, Daisuke Ioka, Hiroyuki Matsuoka, et al. Bacteria expressing single-chain immunotoxin inhibit malaria parasite development in mosquitoes [J]. Molecular & Biochemical Parasitology 2001,113:89-96.
[33]倪剑锋,纪剑飞,吕安国等.抗GD2单链抗体-IL-2融合蛋白基因的构建及表达[J].生物医学工程学杂志,2007,24(1)：170-175.
[34]朱毅,朱进,冯振卿等.日本血吸虫单克隆抗抗独特型抗体NP48单特异性双链抗体的构建、表达与初步鉴定[J].中国血吸虫病防治杂志,2005,17(4)：241-245.
[35]倪剑锋,纪剑飞,白向阳等.抗GD2抗CD16单链双特异性抗体的构建及在大肠杆菌中的表达[J].生物医学工程学杂志,2007,24(3)：659-663.
[1]陈建国.DNA疫苗佐剂的研究进展[J].中华疾病控制杂志,2009,13(1)：87-90.
[2]葛金玲.白细胞介素-18在DNA疫苗免疫中的佐剂效应[J].中国生物制品学杂志,2007,20(7)：547-9.
[3]任莉莉.IL-18在免疫相关性疾病发病机制中的意义[J].广东医学,2004,25(1)：99-101.
[4]Kenji N, Tomohiro Y, Hiroko T, et al. Interleukin-18 is a unique cytokine that stimulates both Th1 and Th2 responses depending on its cytokine milieu[J]. Cytokine Gromthc Factor Rev,2001,12(1):53-72.
[5]Feng Wei, Quan Liu, Yu-jia Zha, et al. IL-18 enhances protective effect in mice immunized with a Schistosoma japonicum FABP DNA vaccine [J]. Acta Tropica, 2009,111:284-8.
[6]Feng Wei, Quan Liu, Sheng-yan Gao, et al. Enhancement by IL-18 of the protective effect of a Schistosoma japonicum 26 kDa GST plasmid DNA vaccine in mice [J]. Vaccine,2008,26:4145-9.
[7]Dupre L,Kremer L,Wolowezuk I,et al. Immunostimulatory effect of IL-18-encoding plasmid in DNA vaccination against murine Schistosoma mansoni infection[J]. Vaccine,2001,19(11-12):1373-80.
[8]Esther T,Eva PJ,Laura LF,et al. The combination of DNA vectors expressing IL-12+IL-18 elicits high protective immune response against cutaneous leishmaniasis after priming with DNA-p36/LACK and the cytokines followed by a booster with a vaccine virus recombinant expressing p36/LACK [J]. Microb Infect,2003,5(1):73-84.
[9]H. Singha, A.I. Mallick, C. Jana, et al. Escheriosomes entrapped DNA vaccine co-expressing Cu-Zn superoxide dismutase and IL-18 confers protection against Brucella abortus [J]. Microbes and Infection,2008,10:1089-96.
[10]Seung H. Kim, Daeho Cho, Tae S. Kim. Anti-CD3 single-chain Fv/interleukin-18 fusion DNA induces anti-mycobacterial resistance via efficient interferon-γ production in BALB/c mice infected with Mycobacterium avium [J]. Vaccine, 2006,24:3365-73.
[11]Triccas JA, Sun L, Palendira U, et al. Comparative affects of plasmid-encoded interleukin 12 and interleukin 18 on the protective efficacy of DNA vaccination against Mycobacterium tuberculosis[J]. Immunol Cell Biol,2002,80(4):346-50.
[12]Jian-zhong chen, Hai-hong zhu, Ke-zhou liu,et al. Enhancing cellular immune response to HBV M DNA vaccine in mice by codelivery of interleukin-18 recombinant[J]. Journal of Zhejiang University SCIENCE,2004,5(4):467-71.
[13]吴欣,黄祖瑚,成军,等.IL-12和IL-18基因免疫对HBcAg核酸疫苗诱导小鼠(H-2d)特异性免疫应答的影响[J].中国免疫学杂志,2007,23：103-7.
[14]Ming-xiao Ma, Ning-yi Jin, Zhen-guo Wang,et al. Construction and immunogenicity of recombinant fowlpox vaccines coexpressing HA of AIV H5N1 and chicken IL18 [J]. Vaccine,2006,24:4304-11.
[15]Ming-xiao Ma, Ning-yi Jin, Guo-shun Shen,et al. Immune responses of swine inoculated with a recombinant fowlpox virus co-expressing P12A and 3C of FMDV and swine IL-18 [J]. Veterinary Immunology and Immunopathology 2008, 121:1-7.
[16]Ming-xiao Ma, Ning-yi Jin, Hui Juan Liu, et al. Immunogenicity of plasmids encoding P12A and 3C of FMDV and swine IL-18[J]. Antiviral Research,2007, 76:59-67.
[17]Xi-Ju Shi, Bin Wang, Ming Wang. Immune enhancing effects of recombinant bovine IL-18 on-foot-and-mouth disease vaccination in mice model [J]. Vaccine, 2007,25:1257-64.
[18]沈国顺,金宁一,秦晓光,等.表达PRRS病毒GP5、GP3和猪IL-18的核酸疫苗的构建及实验免疫研究[J]. 免疫学杂志,2006,22(6)：629-34.
[19]葛金玲,贺冰,袁静,等.IL-18基因佐剂协同HSV-1 gB DNA疫苗免疫诱导的特异性免疫应答[J].中国免疫学杂志,2007,23：317-9.
[20]唐梦君,王红宁,周生,等.IBV M基因与IL-18基因共表达DNA疫苗的免疫原性[J].中国兽医学报,2008,28(7)：757-761.
[21]胡慧琼,王红宁,周生,等.山地乌骨鸡IL-2、IL-15、IL-18及IFN-γ真核表达载体构建及其对IBV DNA疫苗免疫增强作用研究[J].高科技通讯,2006,16(10)：1056-62.
[22]余夏萌,寿春波,章晓栋,等.鸡IL-18对IBDV多聚蛋白DNA疫苗的免疫增强作用研究[J].浙江大学学报,2008,34(1)：13-8.
[23]Billaut-Mulot O,Idziorek T, Loyens MJ,et al. Modulation cellular and humoral immune reponses to a multiepitopic HIV-1 DNA vaccine by interleukin-18 DNA immunization/virla protein boost[J]. Vaccine,2001,19(20-22):2803-11.
[24]Hanlon L, Argyle D, Bain D, et al. Feline leukemia virus DNA vaccine efficacy is enhanced by coadministration with interleukine-12(IL-12) and IL-18 expression vectors[J]. Virology,2001,75(18):8424-33.
[2]Chitsulo L, Engels D, Montresor A, et al. The global status of schistosomiasis and its control[J]. Acta Trop,2000,77(1):41-51.
[3]McManus DP, Loukas A. Current status of vaccines for schistosomiasis[J]. Clin Microbiol Rev,2008,21(1):225-242.
[4]Prevention and control of schistosomiasis and soil-transmitted helminthiasis[J]. World Health Organ Tech Rep Ser,2002,912:i-vi,1-57, back cover.
[5]King CH, Dickman K, Tisch DJ. Reassessment of the cost of chronic helmintic infection:a meta-analysis of disability-related outcomes in endemic schistosomiasis[J]. Lancet,2005,365(9470):1561-1569.
[6]Zhou XN, Guo JG, Wu XH, et al. Epidemiology of schistosomiasis in the People's Republic of China,2004[J]. Emerg Infect Dis,2007,13(10): 1470-1476.
[7]郝阳,郑洁,朱蓉,等.2009年全国血吸虫病疫情通报[J].中国血吸虫病防治杂志,2010,22：521-527.
[8]汪世平主编.医学寄生虫学[M].北京：高等教育出版社,2009.
[9]Giboda M, Smith JM. Schistosoma mansoni eggs as a target for praziquantel: efficacy of oral application in mice[J]. J Trop Med Hyg,1994,97(2):98-102.
[10]Matsuda H, Tanaka H, Nogami S, et al. Mechanism of action of praziquantel on the eggs of Schistosoma japonicum[J]. Jpn J Exp Med,1983,53(6): 271-274.
[11]陈峰,蔡为民,陈智,等.血吸虫病肝纤维的患者转化生长因子β1mRNA的水平极其临床意义[J].中国寄生虫学与寄生虫病杂志,1999,17(3)：140.
[12]蔡卫民,张立煌,孙永良,等.日本血吸虫病肝纤维化患者免疫调节有关因素的探讨[J].中华传染病杂志,1993,11：63.
[13]Rockey DC. Hepatic fibrosis, stellate cells, and portal hypertension[J]. Clin Liver Dis,2006,10(3):459-479, vii-viii.
[14]Ramachandran P, Iredale JP. Reversibility of liver fibrosis[J]. Ann Hepatol, 2009,8(4):283-291.
[15]Chiang DJ, Pritchard MT, Nagy LE. Obesity, diabetes mellitus, and liver fibrosis[J]. Am J Physiol Gastrointest Liver Physiol,2011,300(5):G697-702.
[16]Bartley PB, Ramm GA, Jones MK, et al. A contributory role for activated hepatic stellate cells in the dynamics of Schistosoma japonicum egg-induced fibrosis[J]. Int J Parasitol,2006,36(9):993-1001.
[17]Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis[J]. J Gastroenterol Hepatol,2007,22 Suppl 1:S79-84.
[18]Chu D, Li C, Wu Q, et al. Paeoniflorin prevents hepatic fibrosis of Schistosomiasis japonica by inhibiting TGF-β1 production from macrophages in mice[J]. Frontiers of Medicine in China,2008,2(2):154-165.
[19]Yang J, Zheng J, Wu L, et al. NDRG2 ameliorates hepatic fibrosis by inhibiting the TGF-betal/Smad pathway and altering the MMP2/TIMP2 ratio in rats[J]. PLoS One,2011,6(11):e27710.
[20]Yoshida K, Matsuzaki K. Differential Regulation of TGF-beta/Smad Signaling in Hepatic Stellate Cells between Acute and Chronic Liver Injuries[J]. Front Physiol,2012,3:53.
[21]Ahmad ZA, Yeap SK, Ali AM, et al. scFv Antibody:Principles and Clinical Application[J]. Clin Dev Immunol,2012,2012:980250.
[22]Tang LX, He RH, Yang G, et al. Asiatic acid inhibits liver fibrosis by blocking TGF-beta/Smad signaling in vivo and in vitro[J]. PLoS One,2012,7(2): e31350.
[23]Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling[J]. Nature,2003,425(6958):577-584.
[24]Pearce EJ, Caspar P, Grzych JM, et al. Downregulation of Thl cytokine production accompanies induction of Th2 responses by a parasitic helminth, Schistosoma mansoni[J]. J Exp Med,1991,173(1):159-166.
[25]Grzych JM, Pearce E, Cheever A, et al. Egg deposition is the major stimulus for the production of Th2 cytokines in murine schistosomiasis mansoni[J]. J Immunol,1991,146(4):1322-1327.
[26]Vella AT, Pearce EJ. CD4+ Th2 response induced by Schistosoma mansoni eggs develops rapidly, through an early, transient, ThO-like stage[J]. J Immunol,1992,148(7):2283-2290.
[27]Kent G, Gay S, Inouye T, et al. Vitamin A-containing lipocytes and formation of type III collagen in liver injury[J]. Proc Natl Acad Sci U S A,1976,73(10): 3719-3722.
[28]Maher JJ, McGuire RF. Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo[J]. J Clin Invest,1990,86(5):1641-1648.
[29]Milani S, Herbst H, Schuppan D, et al. Cellular localization of type Ⅰ Ⅲ and IV procollagen gene transcripts in normal and fibrotic human liver[J]. Am J Pathol,1990,137(1):59-70.
[30]Casini A, Pinzani M, Milani S, et al. Regulation of extracellular matrix synthesis by transforming growth factor beta 1 in human fat-storing cells [J]. Gastroenterology,1993,105(1):245-253.
[31]Chiaramonte MG, Cheever AW, Malley JD, et al. Studies of murine schistosomiasis reveal interleukin-13 blockade as a treatment for established and progressive liver fibrosis[J]. Hepatology,2001,34(2):273-282.
[32]Gressner AM, Weiskirchen R, Breitkopf K, et al. Roles of TGF-beta in hepatic fibrosis[J]. Front Biosci,2002,7:d793-807.
[33]De Bleser PJ, Jannes P, van Buul-Offers SC, et al. Insulinlike growth factor-II/mannose 6-phosphate receptor is expressed on CC14-exposed rat fat-storing cells and facilitates activation of latent transforming growth factor-beta in cocultures with sinusoidal endothelial cells[J]. Hepatology,1995, 21(5):1429-1437.
[34]Horras CJ, Lamb CL, Mitchell KA. Regulation of hepatocyte fate by interferon-gamma[J]. Cytokine Growth Factor Rev,2011,22(1):35-43.
[35]Tao FF, Yang YF, Wang H, et al. Thl-type epitopes-based cocktail PDDV attenuates hepatic fibrosis in C57BL/6 mice with chronic Schistosoma japonicum infection[J]. Vaccine,2009,27(31):4110-4117.
[36]Dooley S, Said HM, Gressner AM, et al. Y-box protein-1 is the crucial mediator of antifibrotic interferon-gamma effects[J]. J Biol Chem,2006, 281(3):1784-1795.
[37]Weng H, Mertens PR, Gressner AM, et al. IFN-gamma abrogates profibrogenic TGF-beta signaling in liver by targeting expression of inhibitory and receptor Smads[J]. J Hepatol,2007,46(2):295-303.
[38]Weng HL, Ciuclan L, Liu Y, et al. Profibrogenic transforming growth factor-beta/activin receptor-like kinase 5 signaling via connective tissue growth factor expression in hepatocytes[J]. Hepatology,2007,46(4): 1257-1270.
[39]Zhang L, Mi J, Yu Y, et al. IFN-gamma gene therapy by intrasplenic hepatocyte transplantation:a novel strategy for reversing hepatic fibrosis in Schistosoma japonicum-infectsd mice[J]. Parasite Immunol,2001,23(1): 11-17.
[40]Ushio S, Namba M, Okura T, et al. Cloning of the cDNA for human IFN-gamma-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein[J]. J Immunol,1996,156(11):4274-4279.
[41]Wei F, Liu Q, Gao S, et al. Enhancement by IL-18 of the protective effect of a Schistosoma japonicum 26kDa GST plasmid DNA vaccine in mice[J]. Vaccine, 2008,26(33):4145-4149.
[42]Wei F, Liu Q, Zhai Y, et al. IL-18 enhances protective effect in mice immunized with a Schistosoma japonicum FABP DNA vaccine[J]. Acta Trop, 2009,111(3):284-288.
[43]Zhang LH, Pan JP, Yao HP, et al. Intrasplenic transplantation of IL-18 gene-modified hepatocytes:an effective approach to reverse hepatic fibrosis in schistosomiasis through induction of dominant Thl response[J]. Gene Ther, 2001,8(17):1333-1342.
[44]Rockey DC, Maher JJ, Jarnagin WR, et al. Inhibition of rat hepatic lipocyte activation in culture by interferon-gamma[J]. Hepatology,1992,16(3): 776-784.
[45]Tiggelman AM, Boers W, Linthorst C, et al. Collagen synthesis by human liver (myo)fibroblasts in culture:evidence for a regulatory role of IL-1 beta, IL-4, TGF beta and IFN gamma[J]. J Hepatol,1995,23(3):307-317.
[46]Baroni GS, D'Ambrosio L, Curto P, et al. Interferon gamma decreases hepatic stellate cell activation and extracellular matrix deposition in rat liver fibrosis[J]. Hepatology,1996,23(5):1189-1199.
[47]Rockey DC, Chung JJ. Interferon gamma inhibits lipocyte activation and extracellular matrix mRNA expression during experimental liver injury: implications for treatment of hepatic fibrosis[J]. J Investig Med,1994,42(4): 660-670.
[48]Toyonaga T, Hino O, Sugai S, et al. Chronic active hepatitis in transgenic mice expressing interferon-gamma in the liver[J]. Proc Natl Acad Sci U S A,1994, 91(2):614-618.
[49]Weisser NE, Hall JC. Applications of single-chain variable fragment antibodies in therapeutics and diagnostics[J]. Biotechnol Adv,2009,27(4): 502-520.
[50]Lee YC, Leu SJ, Hung HC, et al. A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage[J]. Vet Immunol Immunopathol,2007,117(1-2): 75-85.
[51]Das D, Kriangkum J, Nagata LP, et al. Development of a biotin mimic tagged ScFv antibody against western equine encephalitis virus:bacterial expression and refolding[J]. J Virol Methods,2004,117(2):169-177.
[52]Ascione A, Capecchi B, Campitelli L, et al. Human monoclonal antibodies in single chain fragment variable format with potent neutralization activity against influenza virus H5N1[J]. Antiviral Res,2009,83(3):238-244.
[53]Hu S, Li L, Qiao J, et al. Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris[J]. Protein Expr Purif,2006,47(1):249-257.
[54]Walper SA, Anderson GP, Brozozog Lee PA, et al. Rugged Single Domain Antibody Detection Elements for Bacillus anthracis Spores and Vegetative Cells[J]. PLoS One,2012,7(3):e32801.
[55]汪世平,赵慰先,易新元,等.日本血吸虫未成熟虫卵可溶性抗原诱导抗卵 免疫初步研究[J].湖南医科大学学报,1995,20(3)：193-196.
[56]汪世平,赵慰先,周泪波,等.日本血吸虫卵的体外培养与抗卵胚发育研究[J].中华医学杂志,1996,76(3)：218-221.
[57]汪世平,赵慰先,易新元,等.日本血吸虫未成熟卵可溶性抗原的初步分析[J].中国人兽共患病杂志,1995,11(4)：35-37.
[58]汪世平,周泪波,张顺科,等.日本血吸虫未成熟虫卵对小鼠肝肉芽肿形成的免疫学影响[J].中华医学杂志,1997,77(10)：768-770.
[59]汪世平,周泪波,沈国励,等.日本血吸虫未成熟虫卵26/28kDa抗原诱导抗雌虫生殖和抗卵胚发育免疫的研究[J].中国寄生虫学与寄生虫病杂志,1997,15(2)：79-83.
[60]汪世平,周泪波,曾宪芳,等.抗日本血吸虫生殖及卵胚发育免疫的研究[J].中国血吸虫病防治杂志,1998,10：7-12.
[61]汪世平,周泪波,曾宪芳,等.抗日本血吸虫未成熟虫卵部分纯化抗原免疫血清的制备及定位观察[J].中华传染病杂志,1997,15(3)：155-156.
[62]He Z, Wang SP, Xiao XQ, et al. Construction screening and application of single-chain Fv antibody library against immature egg of Schistosoma japonicum[J]. Prog Biochem Biophys,2008,35:921-928.
[63]Kozak M. The scanning model for translation:an update[J]. J Cell Biol,1989, 108(2):229-241.
[64]Stewart CA, Horton R, Allcock RJ, et al. Complete MHC haplotype sequencing for common disease gene mapping[J]. Genome Res,2004,14(6): 1176-1187.
[65]萨姆布克J,拉塞尔WD,黄培堂,等.分子克隆实验指南.第三版[M].北京：科学出版社,2002,8.
[66]Bradbury AR, Marks JD. Antibodies from phage antibody libraries[J]. J Immunol Methods,2004,290(1-2):29-49.
[67]Marasco WA, Haseltine WA, Chen SY. Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody [J]. Proc Natl Acad Sci U S A,1993,90(16):7889-7893.
[68]Biocca S, Pierandrei-Amaldi P, Campioni N, et al. Intracellular immunization with cytosolic recombinant antibodies[J]. Biotechnology (N Y),1994,12(4): 396-399.
[69]Cohen PA. Intrabodies. Targeting scFv expression to eukaryotic intracellular compartments[J]. Methods Mol Biol,2002,178:367-378.
[70]Gao DM, Wang SP, He Z, et al. Schistosoma japonicum:screening of cercariae cDNA library by specific single-chain antibody against SIEA26-28 ku and immunization experiment of the recombinant plasmids containing the selected genes[J]. Parasitol Res,2010,107(1):127-134.
[71]高冬梅.SIEA26-28ku-scFv抗日本血吸虫病治疗性疫苗的免疫靶向作用及其效果的初步观察[D].长沙：中南大学,2010.
[72]Heuser C, Guhlke S, Matthies A, et al. Anti-CD30-scFv-Fc-IL-2 antibody-cytokine fusion protein that induces resting NK cells to highly efficient cytolysis of Hodgkin's lymphoma derived tumour cells[J]. Int J Cancer,2004,110(3):386-394.
[73]Halin C, Gafner V, Villani ME, et al. Synergistic therapeutic effects of a tumor targeting antibody fragment, fused to interleukin 12 and to tumor necrosis factor alpha[J]. Cancer Res,2003,63(12):3202-3210.
[74]Helguera G, Dela Cruz JS, Lowe C, et al. Vaccination with novel combinations of anti-HER2/neu cytokines fusion proteins and soluble protein antigen elicits a protective immune response against HER2/neu expressing tumors[J]. Vaccine,2006,24(3):304-316.
[75]Higuchi R, Krummel B, Saiki RK. A general method of in vitro preparation and specific mutagenesis of DNA fragments:study of protein and DNA interactions[J]. Nucleic Acids Res,1988,16(15):7351-7367.
[76]Lu Q. Seamless cloning and gene fusion[J]. Trends Biotechnol,2005,23(4): 199-207.
[77]Gustavsson M, Lehtio J, Denman S, et al. Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris[J]. Protein Eng,2001,14(9):711-715.
[78]Huston JS, Tai MS, McCartney J, et al. Antigen recognition and targeted delivery by the single-chain Fv[J]. Cell Biophys,1993,22(1-3):189-224.
[79]Arnold K, Bordoli L, Kopp J, et al. The SWISS-MODEL workspace:a web-based environment for protein structure homo logy modelling[J]. Bioinformatics,2006,22(2):195-201.
[80]Schwede T, Kopp J, Guex N, et al. SWISS-MODEL:An automated protein homology-modeling server[J]. Nucleic Acids Res,2003,31(13):3381-3385.
[81]Schwede T, Diemand A, Guex N, et al. Protein structure computing in the genomic era[J]. Res Microbiol,2000,151(2):107-112.
[82]Bazan JF, Timans JC, Kastelein RA. A newly defined interleukin-1?[J]. Nature, 1996,379(6566):591.
[83]Marchler-Bauer A, Lu S, Anderson JB, et al. CDD:a Conserved Domain Database for the functional annotation of proteins [J]. Nucleic Acids Res,2011, 39(Database issue):D225-229.
[84]Marchler-Bauer A, Bryant SH. CD-Search:protein domain annotations on the fly[J]. Nucleic Acids Res,2004,32(Web Server issue):W327-331.
[85]Marchler-Bauer A, Anderson JB, Chitsaz F, et al. CDD:specific functional annotation with the Conserved Domain Database[J]. Nucleic Acids Res,2009, 37(Database issue):D205-210.
[86]Okamura H, Tsutsi H, Komatsu T, et al. Cloning of a new cytokine that induces IFN-gamma production by T cells[J]. Nature,1995,378(6552):88-91.
[87]Wigler M, Silverstein S, Lee LS, et al. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells[J]. Cell,1977,11(1):223-232.
[88]Sun CJ, Pan SP, Xie QX, et al. Preparation of chitosan-plasmid DNA nanoparticles encoding zona pellucida glycoprotein-3alpha and its expression in mouse[J]. Mol Reprod Dev,2004,68(2):182-188.
[89]Huntley JF, Stabel JR, Paustian ML, et al. Expression library immunization confers protection against Mycobacterium avium subsp. paratuberculosis infection[J]. Infect Immun,2005,73(10):6877-6884.
[90]Duran EM, Shapshak P, Worley J, et al. Presenilin-1 detection in brain neurons and FOXP3 in peripheral blood mononuclear cells:normalizer gene selection for real time reverse transcriptase pcr using the deltadeltaCt method[J]. Front Biosci,2005,10:2955-2965.
[91]Matsuoka M, Tsukamoto H. Stimulation of hepatic lipocyte collagen production by Kupffer cell-derived transforming growth factor beta: implication for a pathogenetic role in alcoholic liver fibrogenesis[J]. Hepatology,1990,11(4):599-605.
[92]Bachem MG, Meyer D, Melchior R, et al. Activation of rat liver perisinusoidal lipocytes by transforming growth factors derived from myofibroblastlike cells. A potential mechanism of self perpetuation in liver fibrogenesis[J]. J Clin Invest,1992,89(1):19-27.
[93]Bissell DM, Wang SS, Jarnagin WR, et al. Cell-specific expression of transforming growth factor-beta in rat liver. Evidence for autocrine regulation of hepatocyte proliferation[J]. J Clin Invest,1995,96(1):447-455.
[94]Bachem MG, Riess U, Melchior R, et al. Transforming growth factors (TGF alpha and TGF beta 1) stimulate chondroitin sulfate and hyaluronate synthesis in cultured rat liver fat storing cells[J]. FEBS Lett,1989,257(1):134-137.
[95]Qi Z, Atsuchi N, Ooshima A, et al. Blockade of type beta transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat[J]. Proc Natl Acad Sci U S A,1999,96(5):2345-2349.
[96]George J, Roulot D, Koteliansky VE, et al. In vivo inhibition of rat stellate cell activation by soluble transforming growth factor beta type II receptor:a potential new therapy for hepatic fibrosis[J]. Proc Natl Acad Sci U S A,1999, 96(22):12719-12724.
[97]Yata Y, Gotwals P, Koteliansky V, et al. Dose-dependent inhibition of hepatic fibrosis in mice by a TGF-beta soluble receptor:implications for antifibrotic therapy[J]. Hepatology,2002,35(5):1022-1030.
[98]Zhang BB, Jiao YW, Cai WM, et al. Influence of interferon gamma treatment on expression of TGF-betal and its receptors in liver fibrosis of mice with schistosomiasis japonica[J]. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi,2004,22(6):340-343.
[99]Paiva LA, Maya-Monteiro CM, Bandeira-Melo C, et al. Interplay of cysteinyl leukotrienes and TGF-beta in the activation of hepatic stellate cells from Schistosoma mansoni granulomas[J]. Biochim Biophys Acta,2010,1801(12): 1341-1348.
[100]Chu D, Luo Q, Li C, et al. Paeoniflorin inhibits TGF-beta1-mediated collagen production by Schistosoma japonicum soluble egg antigen in vitro [J]. Parasitology,2007,134(Pt 11):1611-1621.
[101]Li J, Wang W, Shen JL. The role of TGFbetal and IL-13 in cellular signal transduction of hepatic fibrosis of schistosomiasis[J]. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi,2009,27(4):357-360.
[102]Zhang JP, Zhang M, Jin C, et al. Matrine inhibits production and actions of fibrogenic cytokines released by mouse peritoneal macrophages[J]. Acta Pharmacol Sin,2001,22(8):765-768.
[103]Friedman SL. Hepatic Fibrosis. In:Schiff ER, Sorrell MF, Maddrey WC, eds. Schiff's Diseases of the Liver[J]. Lippincott Williams & Wilkins,2003: 409-427.
[104]Eng FJ, Friedman SL. Fibrogenesis I. New insights into hepatic stellate cell activation:the simple becomes complex[J]. Am J Physiol Gastrointest Liver Physiol,2000,279(1):G7-G11.
[105]Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury[J]. J Biol Chem,2000,275(4):2247-2250.
[106]Darwich L, Coma G, Pena R, et al. Secretion of interferon-gamma by human macrophages demonstrated at the single-cell level after costimulation with interleukin (IL)-12 plus IL-18[J]. Immunology,2009,126(3):386-393.
[107]Bansal R, Prakash J, de Ruijter M, et al. Peptide-modified albumin carrier explored as a novel strategy for a cell-specific delivery of interferon gamma to treat liver fibrosis[J]. Mol Pharm,2011,8(5):1899-1909.
[108]Ha MH, Wei L, Rao HY, et al Effect of interferon-gamma on hepatic stellate cells stimulated by acetaldehyde[J]. Hepatogastroenterology,2008,55(84): 1059-1065.
[109]Schmitt-Graff A, Kruger S, Bochard F, et al. Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers[J]. Am J Pathol,1991,138(5):1233-1242.
[110]Carpino G, Morini S, Ginanni Corradini S, et al Alpha-SMA expression in hepatic stellate cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis after liver transplantation[J]. Dig Liver Dis, 2005,37(5):349-356.
[111]Iwaisako K, Brenner DA, Kisseleva T. What's new in liver fibrosis? The origin of myofibroblasts in liver fibrosis[J]. J Gastroenterol Hepatol,2012,27 Suppl 2:65-68.
[112]Qu Y, Chen WH, Zong L, et al.18alpha-Glycyrrhizin induces apopto sis and suppresses activation of rat hepatic stellate cells [J]. Med Sci Monit,2012, 18(1):BR24-32.
[113]Ping J, Gao AM, Qin HQ, et al. Indole-3-carbinol enhances the resolution of rat liver fibrosis and stimulates hepatic stellate cell apopto sis by blocking the inhibitor of kappaB kinase alpha/inhibitor of kappaB-alpha/nuclear factor-kappaB pathway[J]. J Pharmacol Exp Ther,2011,339(2):694-703.
[114]Friedman SL, Roll FJ, Boyles J, et al Hepatic lipocytes:the principal collagen-producing cells of normal rat liver[J]. Proc Natl Acad Sci U S A, 1985,82(24):8681-8685.
[115]Bedossa P, Paradis V. Approaches for treatment of liver fibrosis in chronic hepatitis C[J]. Clin Liver Dis,2003,7(1):195-210.
[116]Roderfeld M, Hemmann S, Roeb E. Mechanisms of fibrinolysis in chronic liver injury (with special emphasis on MMPs and TIMPs)[J]. Z Gastroenterol, 2007,45(1):25-33.
[117]Murphy FR, Issa R, Zhou X, et al. Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of metalloproteinase-1 is mediated via effects on matrix metalloproteinase inhibition:implications for reversibility of liver fibrosis[J]. J Biol Chem,2002,277(13):11069-11076.
[118]Fowell AJ, Collins JE, Duncombe DR, et al Silencing tissue inhibitors of metalloproteinases (TIMPs) with short interfering RNA reveals a role for TIMP-1 in hepatic stellate cell proliferation[J]. Biochem Biophys Res Commun,2011,407(2):277-282.
[119]Fabre V, Wu H, PondTor S, et al. Tissue inhibitor of matrix-metalloprotease-1 predicts risk of hepatic fibrosis in human Schistosoma japonicum infection[J]. J Infect Dis,2011,203(5):707-714.
[120]Tian Z, Wang SP, Zeng SH, et al. Characterization and vaccination of two novel Schistosoma japonicum genes screened from a cercaria cDNA library[J]. Parasitol Res,2012,110(1):403-409.
[121]Cheever AW, Duvall RH, Hallack TA, Jr., et al. Variation of hepatic fibrosis and granuloma size among mouse strains infected with Schistosoma mansoni[J]. Am J Trop Med Hyg,1987,37(1):85-97.
[122]Kofta W, Wedrychowicz H. c-DNA vaccination against parasitic infections: advantages and disadvantages[J]. Vet Parasitol,2001,100(1-2):3-12.
[123]Dinarello CA, Fantuzzi G. Interleukin-18 and host defense against infection[J]. J Infect Dis,2003,187 Suppl 2:S370-384.
[124]Hou X, Yu F, Man S, et al. Negative regulation of Schistosoma japonicum egg-induced liver fibrosis by natural killer cells [J]. PLoS Negl Trop Dis,2012, 6(1):e1456.
[125]Pearce EJ, MacDonald AS. The immunobiology of schistosomiasis[J]. Nat Rev Immunol,2002,2(7):499-511.
[126]Kim HJ, Song SB, Choi JM, et al. IL-18 downregulates collagen production in human dermal fibroblasts via the ERK pathway[J]. J Invest Dermatol,2010, 130(3):706-715.
[127]Arthur MJ. Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis[J]. Am J Physiol Gastrointest Liver Physiol,2000,279(2):G245-249.
[128]McCrudden R, Iredale JP. Liver fibrosis, the hepatic stellate cell and tissue inhibitors of metalloproteinases [J]. Histol Histopathol,2000,15(4): 1159-1168.
[129]Singh KP, Gerard HC, Hudson AP, et al. Expression of matrix metalloproteinases and their inhibitors during the resorption of schistosome egg-induced fibrosis in praziquantel-treated mice[J]. Immunology,2004, 111(3):343-352.
[130]Rhyner C, Konthur Z, Blaser K, et al. High-throughput isolation of recombinant antibodies against recombinant allergens[J]. Biotechniques,2003, 35(4):672-674.
[131]Menzel C, Schirrmann T, Konthur Z, et al. Human antibody RNase fusion protein targeting CD30+ lymphomas[J]. Blood,2008,111(7):3830-3837.
[132]Kim SH, Cho D, Kim TS. Anti-CD3 single-chain Fv/interleukin-18 fusion DNA induces anti-myco bacterial resistance via efficient interferon-gamma production in BALB/c mice infected with Mycobacterium avium[J]. Vaccine, 2006,24(16):3365-3373.
[133]Gillies SD, Young D, Lo KM, et al Biological activity and in vivo clearance of antitumor antibody/cytokine fusion proteins[J]. Bioconjug Chem,1993, 4(3):230-235.
[1]Matthias Peipp, Domenica Saul, Karin Barbin, et al. Efficient eukaryotic expression of fluorescent scFv fusion proteins directed against CD antigens for FACS applications [J]. Journal of Immunological Methods,2004, 285:265-280.
[2]Nina E. Weisser, J. Christopher Hall. Applications of single-chain variable fragment antibodies in therapeutics and diagnostics [J]. Biotechnology Advances,2009,27:502-520.
[3]KurtC. Almquist, Michael D. McLean, Yongqing Niu, et al. Expression of an anti-botulinum toxin A neutralizing single-chain Fv recombinant antibody in transgenic tobacco [J]. Vaccine,2006,24:2079-2086.
[4]Yu-Ching Lee, Sy-Jye C. Leu, Han-Chang Hung, et al. A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage [J]. Veterinary Immunology and Immunopathology,2007,117:75-85.
[5]D. Das, J. Kriangkum, L.P. Nagata, et al. Development of a biotin mimic tagged ScFv antibody against western equine encephalitis virus:bacterial expression and refolding [J]. Journal of Virological Methods,2004, 117:169-177.
[6]Alessandro Ascione, Barbara Capecchi, Laura Campitelli, et al. Human monoclonal antibodies in single chain fragment variable format with potent neutralization activity against influenza virus H5N1 [J]. Antiviral Research, 2009,2494:1-7.
[7]赵泽文,梁志清,史常旭等.抗rhEndoglin单链抗体的可溶性表达、纯化及活性鉴定[J].免疫学杂志,2009,25(1)：56-60.
[8]张桂红,刘彦仿,付勇等.抗肝癌单链抗体免疫脂质体的制备及其体外抑瘤实验[J].医学研究生学报,2006,19(1)：3-5.
[9]Siyi Hu, Liangwei Li, Jingjuan Qiao, et al. Codon optimization,expression,and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris [J]. Protein Expression and Purication,2006,47:249-257.
[10]祝怀平,王迎春,季顺东等.抗vWF与GPIb2IX结合部位的噬菌体呈现型单链抗体制备及功能研究[J].中国免疫学杂,2005,21：308-402.
[11]郭安,尹继刚,向梅等. 微小隐孢子虫子孢子与宿主黏附相关蛋白的筛 选[J].中国寄生虫学与寄生虫病杂志,2009,27(1)：87-88.
[12]L.-N. HOE, K.-L. WAN, S. NATHAN.Construction and characterization of recombinant a single-chain variable fragment antibodies against Toxoplasma gondii MIC2 protein [J]. Parasitology 2005,131:759-768.
[13]Jorge Sepulveda, Charles B. Shoemaker. Design and testing of PCR primers for the construction of scFv libraries representing the immunoglobulin repertoire of rats [J]. Journal of Immunological Methods,2008,332:92-102.
[14]Susana Re'fega, Marianne Cluzeaud, Pierre Pe'ry, et al. Production of a functional chicken single-chain variable fragment antibody derived from caecal tonsils B lymphocytes against macrogamonts of Eimeria tenella [J]. Veterinary Immunology and Immunopathology,2004,97:219-230
[15]司进,朱荫昌,曹利民等.抗弓形虫主要表面抗原1单链抗体的筛选及鉴定[J].中国血吸虫病防止杂志,2005,17(3)：202-206.
[16]陈新华,温浩,刘辉等.抗棘球蚴人源单链可变区抗体抑制人体包虫原头节的初步实验[J].中华微生物学和免疫学杂志,2004,24(9)：699-702.
[17]Heather Griffin, Robert Elston, Deborah Jackson, et al. Inhibition of Papillomavirus Protein Function in Cervica Cancer Cells by Intrabody Targeting [J]. J. Mol. Biol. 2006,355:360-378.
[18]Gregory P. White, Els N.T. Meeusen, Susan E. Newton. A single-chain variable region immunoglobulin library from the abomasal lymph node of sheep infected with the gastrointestinal nematode parasite Haemonchus contortus [J]. Veterinary Immunology and Immunopathology,2001, 78:117-129.
[19]Shigeto Yoshida, Hiroyuki Matsuoka, Enjie Luo, et al. A single-chain antibody fragment specific for the Plasmodium berghei ookinete protein Pbs21 confers transmission blockade in the mosquito midgut [J]. Molecular and Biochemical Parasitology,1999,104:195-204.
[20]So-Hee Kim, Seung-Young Hwang, Yong-Seok Lee, et al. Single-Chain Antibody Fragment Specific for Plasmodium vivax Duff Binding Protein [J]. CLINICAL AND VACCINE IMMUNOLOGY,2007,14(6):726-731.
[21]David R. Maass, Gavin B.L. Harrison, Warwick N. Grant, et al. Three surface antigens dominate the mucosal antibody response to gastrointestinal L3-stage strongylid nematodes in field immune sheep [J]. International Journal for Parasitology,2007,37:953-962.
[22]Guilherme Rocha Lino De Souza, Carlos Roberto Prudencio, Rone Cardoso, et al. Construction of an antibodies library(scFv) for selection and characterization of Boophilus microplus antigens [J]. Abstracts/Veterinary Immunology and Immunopathology,2009,128:211-347.
[23]何卓,汪世平,周帅锋等. 抗日本血吸虫生殖功能分子SIEA26-28kDa单链抗体与EGFP的融合表达及靶向性研究[J].中国人兽共患病学报,2008,24(8)：704-707.
[24]何卓,汪世平,肖小芹等.日本血吸虫未成熟卵单链抗体库的构建、筛选及初步应用[J].生物化学与生物物理进展,2008,35(8)：921-928.
[25]Petra Pavlickova, E. Marion Schneider, Hubert Hug. Advances in recombinant antibody microarrays [J]. Clinica Chimica Acta,2004,343:17-35.
[26]CHEN Dai-xiong, HE Ai, ZHAN Xi-mei, et al. Schistosoma japonicum: construction of phage display antibody library and its application in the immunodiagnosis of infection [J]. Chinese Medical Journal,2004, 117(11):1697-1703.
[27]尹继刚,张西臣,李建华等.抗隐孢子虫子孢子ScFv-PE40免疫毒素表达质粒的构建及其在大肠杆菌中的表达[J].寄生虫与医学昆虫学报,2005,12(3)：139-142.
[28]刘晓峰,李淑红,张西臣等.抗隐孢子虫ScFv-PE重组毒素原核表达载体的构建及其对虫体的杀灭作用[J].热带医学杂志,2007,7(4)：307-310.
[29]司进,朱荫昌,曹利民等.弓形虫主要表面抗原1单链抗体与绿色荧光蛋白的融合表达及其生物活性初步观察[J].中国寄生虫学与寄生虫病杂志,2006,24(3)：161-165.
[30]丁雪冰,陈晓光.恶性疟原虫环子孢子蛋白单链抗体与按蚊cecropin A融合基因的构建、原核表达及生物学活性的初步分析[J].寄生虫与医学昆虫学报,2008,15(1)：8-13.
[31]司进,朱荫昌,曹利民等.抗弓形虫靶向抗菌肽的构建及其效应的初步评价[J].中国血吸虫病防治杂志,2005,17(5)：356-361.
[32]Shigeto Yoshida, Daisuke Ioka, Hiroyuki Matsuoka, et al. Bacteria expressing single-chain immunotoxin inhibit malaria parasite development in mosquitoes [J]. Molecular & Biochemical Parasitology 2001,113:89-96.
[33]倪剑锋,纪剑飞,吕安国等.抗GD2单链抗体-IL-2融合蛋白基因的构建及表达[J].生物医学工程学杂志,2007,24(1)：170-175.
[34]朱毅,朱进,冯振卿等.日本血吸虫单克隆抗抗独特型抗体NP48单特异性双链抗体的构建、表达与初步鉴定[J].中国血吸虫病防治杂志,2005,17(4)：241-245.
[35]倪剑锋,纪剑飞,白向阳等.抗GD2抗CD16单链双特异性抗体的构建及在大肠杆菌中的表达[J].生物医学工程学杂志,2007,24(3)：659-663.
[1]陈建国.DNA疫苗佐剂的研究进展[J].中华疾病控制杂志,2009,13(1)：87-90.
[2]葛金玲.白细胞介素-18在DNA疫苗免疫中的佐剂效应[J].中国生物制品学杂志,2007,20(7)：547-9.
[3]任莉莉.IL-18在免疫相关性疾病发病机制中的意义[J].广东医学,2004,25(1)：99-101.
[4]Kenji N, Tomohiro Y, Hiroko T, et al. Interleukin-18 is a unique cytokine that stimulates both Th1 and Th2 responses depending on its cytokine milieu[J]. Cytokine Gromthc Factor Rev,2001,12(1):53-72.
[5]Feng Wei, Quan Liu, Yu-jia Zha, et al. IL-18 enhances protective effect in mice immunized with a Schistosoma japonicum FABP DNA vaccine [J]. Acta Tropica, 2009,111:284-8.
[6]Feng Wei, Quan Liu, Sheng-yan Gao, et al. Enhancement by IL-18 of the protective effect of a Schistosoma japonicum 26 kDa GST plasmid DNA vaccine in mice [J]. Vaccine,2008,26:4145-9.
[7]Dupre L,Kremer L,Wolowezuk I,et al. Immunostimulatory effect of IL-18-encoding plasmid in DNA vaccination against murine Schistosoma mansoni infection[J]. Vaccine,2001,19(11-12):1373-80.
[8]Esther T,Eva PJ,Laura LF,et al. The combination of DNA vectors expressing IL-12+IL-18 elicits high protective immune response against cutaneous leishmaniasis after priming with DNA-p36/LACK and the cytokines followed by a booster with a vaccine virus recombinant expressing p36/LACK [J]. Microb Infect,2003,5(1):73-84.
[9]H. Singha, A.I. Mallick, C. Jana, et al. Escheriosomes entrapped DNA vaccine co-expressing Cu-Zn superoxide dismutase and IL-18 confers protection against Brucella abortus [J]. Microbes and Infection,2008,10:1089-96.
[10]Seung H. Kim, Daeho Cho, Tae S. Kim. Anti-CD3 single-chain Fv/interleukin-18 fusion DNA induces anti-mycobacterial resistance via efficient interferon-γ production in BALB/c mice infected with Mycobacterium avium [J]. Vaccine, 2006,24:3365-73.
[11]Triccas JA, Sun L, Palendira U, et al. Comparative affects of plasmid-encoded interleukin 12 and interleukin 18 on the protective efficacy of DNA vaccination against Mycobacterium tuberculosis[J]. Immunol Cell Biol,2002,80(4):346-50.
[12]Jian-zhong chen, Hai-hong zhu, Ke-zhou liu,et al. Enhancing cellular immune response to HBV M DNA vaccine in mice by codelivery of interleukin-18 recombinant[J]. Journal of Zhejiang University SCIENCE,2004,5(4):467-71.
[13]吴欣,黄祖瑚,成军,等.IL-12和IL-18基因免疫对HBcAg核酸疫苗诱导小鼠(H-2d)特异性免疫应答的影响[J].中国免疫学杂志,2007,23：103-7.
[14]Ming-xiao Ma, Ning-yi Jin, Zhen-guo Wang,et al. Construction and immunogenicity of recombinant fowlpox vaccines coexpressing HA of AIV H5N1 and chicken IL18 [J]. Vaccine,2006,24:4304-11.
[15]Ming-xiao Ma, Ning-yi Jin, Guo-shun Shen,et al. Immune responses of swine inoculated with a recombinant fowlpox virus co-expressing P12A and 3C of FMDV and swine IL-18 [J]. Veterinary Immunology and Immunopathology 2008, 121:1-7.
[16]Ming-xiao Ma, Ning-yi Jin, Hui Juan Liu, et al. Immunogenicity of plasmids encoding P12A and 3C of FMDV and swine IL-18[J]. Antiviral Research,2007, 76:59-67.
[17]Xi-Ju Shi, Bin Wang, Ming Wang. Immune enhancing effects of recombinant bovine IL-18 on-foot-and-mouth disease vaccination in mice model [J]. Vaccine, 2007,25:1257-64.
[18]沈国顺,金宁一,秦晓光,等.表达PRRS病毒GP5、GP3和猪IL-18的核酸疫苗的构建及实验免疫研究[J]. 免疫学杂志,2006,22(6)：629-34.
[19]葛金玲,贺冰,袁静,等.IL-18基因佐剂协同HSV-1 gB DNA疫苗免疫诱导的特异性免疫应答[J].中国免疫学杂志,2007,23：317-9.
[20]唐梦君,王红宁,周生,等.IBV M基因与IL-18基因共表达DNA疫苗的免疫原性[J].中国兽医学报,2008,28(7)：757-761.
[21]胡慧琼,王红宁,周生,等.山地乌骨鸡IL-2、IL-15、IL-18及IFN-γ真核表达载体构建及其对IBV DNA疫苗免疫增强作用研究[J].高科技通讯,2006,16(10)：1056-62.
[22]余夏萌,寿春波,章晓栋,等.鸡IL-18对IBDV多聚蛋白DNA疫苗的免疫增强作用研究[J].浙江大学学报,2008,34(1)：13-8.
[23]Billaut-Mulot O,Idziorek T, Loyens MJ,et al. Modulation cellular and humoral immune reponses to a multiepitopic HIV-1 DNA vaccine by interleukin-18 DNA immunization/virla protein boost[J]. Vaccine,2001,19(20-22):2803-11.
[24]Hanlon L, Argyle D, Bain D, et al. Feline leukemia virus DNA vaccine efficacy is enhanced by coadministration with interleukine-12(IL-12) and IL-18 expression vectors[J]. Virology,2001,75(18):8424-33.