基于家蚕杆状病毒表达系统表达外源基因研究
摘要
家蚕杆状病毒表达系统作为一种真核表达系统,表达的蛋白具有折叠和糖基化,近于真实构相,表达量较大,而且能够可溶性地表达一些原核系统难以表达的大分子量蛋白。我们首先使用家蚕杆状病毒表达系统表达了Mn-SOD基因,并通过低温冷冻真空干燥,以SOD全蚕粉的形式,保持了SOD的活性。对SOD全蚕粉进行了以《保健食品检验与评价技术规范》为标准的安全性和功能性检验。经对小鼠进行人体推荐量360倍的急性毒性检验、90天的长期性毒性检验,小鼠生长正常,各器官无异常现象发生；SOD全蚕粉对ConA刺激导致的淋巴细胞转化,在体外法实验中,显示能明显增加淋巴细胞转化率,提示SOD全蚕粉具有免疫调节作用；经口给予小鼠不同剂量的SOD全蚕粉30天,能提高小鼠迟发型变态反应能力,在高剂量组达到显著水平,但与阳性对照(黄芪组)有较明显的差距。SOD全蚕粉促进小鼠的体重增长,对脾脏生长无影响,但明显增加小鼠的胸腺／体重比值；用SRBC免疫小鼠后,产生抗SRBC抗体,SOD全蚕粉对血凝素也有明显的作用,表明SOD全蚕粉对正常机体的体液免疫功能均有增强作用；经对小鼠巨噬细胞的功能实验,SOD全蚕粉提高了小鼠巨噬细胞吞噬鸡红细胞的能力。
在家蚕体内表达SOD基因后,经冷冻干燥制得SOD全蚕粉,设三个剂量组和阴性、阳性对照组给小鼠灌胃；经口给予小鼠不同剂量的SOD全蚕粉30天,能增加小鼠血和肝脏中的超氧化物歧化酶和谷胱甘肽过氧化物酶的活性,降低小鼠肝脏中丙二醛含量。
在家蚕体内表达SOD基因后,经冷冻干燥制得SOD全蚕粉,小鼠灌胃45天,小鼠尾静脉注稀释的印度墨汁,以吞噬指数表示小鼠碳廓清的能力,碳廓清能力提高,说明加速碳粒的清除,显著提高自然杀伤细胞抗瘤活性,增强机体的非特异性细胞免疫功能。以NIH小鼠为研究模型,观察SOD全蚕粉对NK细胞活性的影响,证实了SOD全蚕粉有明显的能增强NK细胞的活性,从而增强机体的免疫机能。在此基础上,进行抑制肿瘤实验,结果表明提高小鼠抗肿瘤能力,SOD全蚕粉提高S180肿瘤荷瘤小鼠的淋巴细胞转化率、NK细胞活性,抑制小鼠的肿瘤生长；SOD全蚕粉组对肝癌H22的平均抑瘤率为40.3,表现出较强的抗肿瘤活性,能促进T淋巴细胞转化,提高NK细胞活性。
经四氧嘧啶造模成功后小鼠,对其灌胃SOD全蚕粉30天(SOD全蚕粉为家蚕体内表达SOD基因后,经冷冻干燥制得),结果显示SOD全蚕粉能增加小鼠的糖耐量水平,对小鼠的空腹血糖值有降低趋势,达到显著水平。
家蚕杆状病毒表达系统是一个功能强大,可利用范围较广的真核表达系统,然而,由于感染需要节间注射,限制了家蚕杆状病毒表达系统的应用,因此,我们对家蚕杆状病毒表达系统进行了几项攻进。采用荧光增白剂喷洒桑叶,增加家蚕经口对杆状病毒表达系统中携带外源基因病毒的易感性。构建了双表达载体系统,在表达Mn-SOD目的蛋白的同时,恢复表达了多角体基因；多角体基因和Mn-SOD基因被分别插在多角体启动子和P10启动子之下,实现了经口感染。摸索出重组杆状病毒DNA与转染剂混合比例和转染条件,在家蚕体内实现从DNA到病毒的转化,经口感染家蚕幼虫,大大简化外源基因表达的操作手续。
表达的目的蛋白在感染后期常被降解,甚至在感染后期家蚕等昆虫组织器官也受到降解,给目的蛋白的纯化等处理带来许多麻烦,这已成为该表达系统,特别是在产业化开发时遇到的一个严重的问题。为了解决表达蛋白的稳定性。引进和采用了缺少半胱氨酸蛋白酶基因的Bacmid质粒载体(CPD),缺少半胱氨酸蛋白酶和几丁质酶基因的Bacmid质粒载体(CPPD),用这些改进后的Bacmid质粒载体表达外源基因,并在人胰岛素的表达中进行了对比；在猪蓝耳病病毒膜蛋白GP5和M基因表达实验中采用了缺少半胱氨酸蛋白酶基因的Bacmid质粒载体(CPD)。
在家蚕体内表达出人胰岛素,开发出除大肠杆菌、酵母表达系统外,人胰岛素表达的第三条途径。
在家蚕体内表达出猪繁殖与呼吸综合征(蓝耳病)表面抗原蛋白,经ELISA检验有抗原原性,开辟了用家蚕生产DNA等基因工程疫苗的新途径奠定了基础。
Summary
The insect baculovirus expression system is a useful tool for the efficient production of eukaryotic proteins that require correct folding and posttranslational modification such as signal peptide processing, phosphorylation and glycosylation. The baculovirus expression system has been proved to be very efficient for heterologous protein production than that obtained in naturally producing mammalian cells. Superoxide dismutase (SOD) is a metalloenzyme, which catalyzes the conversion of the superoxide radicals into molecular O2 and H2O2 and thus forms a crucial part of the cellular antioxidant defense mechanism. The amount of SOD present in cellular and extracellular environment is crucial for the prevention of disease linked to oxidative stress. We used the silkworm, Bombyx mori, larvae as a bio-reactor and expressed the Mn-SOD by the recombinant bacmid baculoviruses expression system. The fifth instar silkworm larvae expressed SOD 96 h post-infection with recombinant virus (rBacmid/BmNPV/SOD) were collected and dried with a vacuum dryer. To study the function of silkworm larvae powder containing superoxide dismutase and potential practical development, we investigated the safety assessment and effects on immune activity of mice such as the growth of immunity-related organs, delayed-type hypersensitivity (DTH) and charcoal particle clearance ability. The mean body weights in treated mice were significantly heavier than that of the control, meanwhile, the ratio of the thoracic gland/body weight in treated mice was significantly enhanced after 30 days treated with silkworm larvae powder containing manganese superoxide dismutase. The treated mice resulted in a profound activation of the DTH and charcoal particle clearance, and indicated the treated mice have stronger phagocytic activity to exogenous materials. Our data also indicated the feeding treatment was safe with 360 folds of recommended human dosage in acute toxic test. In long-term test, there were no effects of silkworm larvae powder containing SOD on treated mice's growth and inside organs as long as 90 days. Further the electronic microscope investigation showed the intestine, liver, splenocyte and stomach in mice were no obvious changes both in organs and sub-organs such as nucleus, endoplasmic reticulum, mitochondrion, Golgi and peroxisomes after treated for as long as 90 days. We investigated the effects of silkworm larvae powder containing SOD on the immune system of mouse and employed a proteomics approach to examine this phenomenon. Our data on the effects of continuous treatment with SOD-containing silkworm larvae powder showed that the ConA-stimulated splenocyte proliferation of all three treated groups was higher than that of the control. The results of PFC assay also revealed that antibody production was higher in all three treated groups than controlled mice. We investigated the phagocytosis of mouse macrophages. The SOD treatment led to a dose dependent increase of phagocytic activity. We identified six proteins that related to immunity of mice. The data showed all these six matched proteins related immunity presented the increase of expression level in plasma of mouse administrated with silkworm powder including SOD compared to that of control. These findings demonstrate that administration of silkworm larvae powder containing SOD results in enhancement of immunity activities in the mouse. The results also suggested that the SOD expressed in silkworm maybe have potential application in medicine.
We used the bacmid DNA developed in our laboratory for direct injection in silkworm larvae, thereby avoiding the preparation of high-titer viral stocks for infecting larvae. Using silkworms or pupae are 10-100-fold higher than those using Bombyx mori cells or conventional insect cells, indicating that the silkworm or its pupa is one of the most suitable systems for large-scale production of eukaryotic proteins. In this study, we cloned the human insulin gene using human genomic DNA as template, and constructed the recombinant Bacmid DNA including human insulin and FLAG tag. The silkworm larvae were infected with above recombinant Bacmid DNA, and the insulin were purified and identified from the infected worm haemolymph. Several kinds of reBmMNPVbacmid, which are cysteine protease gene deletion (CPD-BmMNPV bacmid) and cysteine protease-and chitinase-deficient (CPPD-BmMNPV bacmid) including human insulin and FLAG tag were constructed. The silkworm larvae were infected with above recombinant Bacmid DNA, and the expressed insulin were purified and identified from the infected worm haemolymph. The highest expression was shown in CPPD-BmMNPV bacmid that is about two times of the wild type of reBmMNPVbacmid, reaching 15.827ng/ml haemolymph. The two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 (E) and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. Two different DNA vaccine constructs, expressing GP5+γ, GP5 and M proteins+γsimultaneously (ORF5/ORF6/y) were constructed. Co-expression of GP5 and M protein in heterodimers can significantly improve the potency of DNA vaccination and could be used as a strategy to develop a new generation of vaccines against PRRSV.
在家蚕体内表达SOD基因后,经冷冻干燥制得SOD全蚕粉,设三个剂量组和阴性、阳性对照组给小鼠灌胃；经口给予小鼠不同剂量的SOD全蚕粉30天,能增加小鼠血和肝脏中的超氧化物歧化酶和谷胱甘肽过氧化物酶的活性,降低小鼠肝脏中丙二醛含量。
在家蚕体内表达SOD基因后,经冷冻干燥制得SOD全蚕粉,小鼠灌胃45天,小鼠尾静脉注稀释的印度墨汁,以吞噬指数表示小鼠碳廓清的能力,碳廓清能力提高,说明加速碳粒的清除,显著提高自然杀伤细胞抗瘤活性,增强机体的非特异性细胞免疫功能。以NIH小鼠为研究模型,观察SOD全蚕粉对NK细胞活性的影响,证实了SOD全蚕粉有明显的能增强NK细胞的活性,从而增强机体的免疫机能。在此基础上,进行抑制肿瘤实验,结果表明提高小鼠抗肿瘤能力,SOD全蚕粉提高S180肿瘤荷瘤小鼠的淋巴细胞转化率、NK细胞活性,抑制小鼠的肿瘤生长；SOD全蚕粉组对肝癌H22的平均抑瘤率为40.3,表现出较强的抗肿瘤活性,能促进T淋巴细胞转化,提高NK细胞活性。
经四氧嘧啶造模成功后小鼠,对其灌胃SOD全蚕粉30天(SOD全蚕粉为家蚕体内表达SOD基因后,经冷冻干燥制得),结果显示SOD全蚕粉能增加小鼠的糖耐量水平,对小鼠的空腹血糖值有降低趋势,达到显著水平。
家蚕杆状病毒表达系统是一个功能强大,可利用范围较广的真核表达系统,然而,由于感染需要节间注射,限制了家蚕杆状病毒表达系统的应用,因此,我们对家蚕杆状病毒表达系统进行了几项攻进。采用荧光增白剂喷洒桑叶,增加家蚕经口对杆状病毒表达系统中携带外源基因病毒的易感性。构建了双表达载体系统,在表达Mn-SOD目的蛋白的同时,恢复表达了多角体基因；多角体基因和Mn-SOD基因被分别插在多角体启动子和P10启动子之下,实现了经口感染。摸索出重组杆状病毒DNA与转染剂混合比例和转染条件,在家蚕体内实现从DNA到病毒的转化,经口感染家蚕幼虫,大大简化外源基因表达的操作手续。
表达的目的蛋白在感染后期常被降解,甚至在感染后期家蚕等昆虫组织器官也受到降解,给目的蛋白的纯化等处理带来许多麻烦,这已成为该表达系统,特别是在产业化开发时遇到的一个严重的问题。为了解决表达蛋白的稳定性。引进和采用了缺少半胱氨酸蛋白酶基因的Bacmid质粒载体(CPD),缺少半胱氨酸蛋白酶和几丁质酶基因的Bacmid质粒载体(CPPD),用这些改进后的Bacmid质粒载体表达外源基因,并在人胰岛素的表达中进行了对比；在猪蓝耳病病毒膜蛋白GP5和M基因表达实验中采用了缺少半胱氨酸蛋白酶基因的Bacmid质粒载体(CPD)。
在家蚕体内表达出人胰岛素,开发出除大肠杆菌、酵母表达系统外,人胰岛素表达的第三条途径。
在家蚕体内表达出猪繁殖与呼吸综合征(蓝耳病)表面抗原蛋白,经ELISA检验有抗原原性,开辟了用家蚕生产DNA等基因工程疫苗的新途径奠定了基础。
Summary
The insect baculovirus expression system is a useful tool for the efficient production of eukaryotic proteins that require correct folding and posttranslational modification such as signal peptide processing, phosphorylation and glycosylation. The baculovirus expression system has been proved to be very efficient for heterologous protein production than that obtained in naturally producing mammalian cells. Superoxide dismutase (SOD) is a metalloenzyme, which catalyzes the conversion of the superoxide radicals into molecular O2 and H2O2 and thus forms a crucial part of the cellular antioxidant defense mechanism. The amount of SOD present in cellular and extracellular environment is crucial for the prevention of disease linked to oxidative stress. We used the silkworm, Bombyx mori, larvae as a bio-reactor and expressed the Mn-SOD by the recombinant bacmid baculoviruses expression system. The fifth instar silkworm larvae expressed SOD 96 h post-infection with recombinant virus (rBacmid/BmNPV/SOD) were collected and dried with a vacuum dryer. To study the function of silkworm larvae powder containing superoxide dismutase and potential practical development, we investigated the safety assessment and effects on immune activity of mice such as the growth of immunity-related organs, delayed-type hypersensitivity (DTH) and charcoal particle clearance ability. The mean body weights in treated mice were significantly heavier than that of the control, meanwhile, the ratio of the thoracic gland/body weight in treated mice was significantly enhanced after 30 days treated with silkworm larvae powder containing manganese superoxide dismutase. The treated mice resulted in a profound activation of the DTH and charcoal particle clearance, and indicated the treated mice have stronger phagocytic activity to exogenous materials. Our data also indicated the feeding treatment was safe with 360 folds of recommended human dosage in acute toxic test. In long-term test, there were no effects of silkworm larvae powder containing SOD on treated mice's growth and inside organs as long as 90 days. Further the electronic microscope investigation showed the intestine, liver, splenocyte and stomach in mice were no obvious changes both in organs and sub-organs such as nucleus, endoplasmic reticulum, mitochondrion, Golgi and peroxisomes after treated for as long as 90 days. We investigated the effects of silkworm larvae powder containing SOD on the immune system of mouse and employed a proteomics approach to examine this phenomenon. Our data on the effects of continuous treatment with SOD-containing silkworm larvae powder showed that the ConA-stimulated splenocyte proliferation of all three treated groups was higher than that of the control. The results of PFC assay also revealed that antibody production was higher in all three treated groups than controlled mice. We investigated the phagocytosis of mouse macrophages. The SOD treatment led to a dose dependent increase of phagocytic activity. We identified six proteins that related to immunity of mice. The data showed all these six matched proteins related immunity presented the increase of expression level in plasma of mouse administrated with silkworm powder including SOD compared to that of control. These findings demonstrate that administration of silkworm larvae powder containing SOD results in enhancement of immunity activities in the mouse. The results also suggested that the SOD expressed in silkworm maybe have potential application in medicine.
We used the bacmid DNA developed in our laboratory for direct injection in silkworm larvae, thereby avoiding the preparation of high-titer viral stocks for infecting larvae. Using silkworms or pupae are 10-100-fold higher than those using Bombyx mori cells or conventional insect cells, indicating that the silkworm or its pupa is one of the most suitable systems for large-scale production of eukaryotic proteins. In this study, we cloned the human insulin gene using human genomic DNA as template, and constructed the recombinant Bacmid DNA including human insulin and FLAG tag. The silkworm larvae were infected with above recombinant Bacmid DNA, and the insulin were purified and identified from the infected worm haemolymph. Several kinds of reBmMNPVbacmid, which are cysteine protease gene deletion (CPD-BmMNPV bacmid) and cysteine protease-and chitinase-deficient (CPPD-BmMNPV bacmid) including human insulin and FLAG tag were constructed. The silkworm larvae were infected with above recombinant Bacmid DNA, and the expressed insulin were purified and identified from the infected worm haemolymph. The highest expression was shown in CPPD-BmMNPV bacmid that is about two times of the wild type of reBmMNPVbacmid, reaching 15.827ng/ml haemolymph. The two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 (E) and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. Two different DNA vaccine constructs, expressing GP5+γ, GP5 and M proteins+γsimultaneously (ORF5/ORF6/y) were constructed. Co-expression of GP5 and M protein in heterodimers can significantly improve the potency of DNA vaccination and could be used as a strategy to develop a new generation of vaccines against PRRSV.
引文
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