摘要
近年来,随着分子生物学、免疫学理论和技术的发展,运用基因重组表达技术研制预防、治疗用生物制品己经成为免疫学领域研究的热点,其中核酸(DNA)疫苗倍受青睐。该疫苗是继灭活疫苗、减毒活疫苗、亚单位疫苗及基因工程疫苗后的新一代疫苗。核酸疫苗具有同时激发机体体液和细胞免疫应答、使用安全、易于生产等优点,并可将多种基因联合在一起制成基因联合疫苗。然而由于DNA疫苗蛋白表达量低,激发的免疫应答较弱,从而限制了其开发应用速度。因此,提高DNA疫苗的免疫效果已成为基因免疫研究中急需解决的问题;目前常采用新型分子佐剂,如白细胞介素、干扰素、胸腺肽和补体分子佐剂等是提高DNA疫苗免疫效果的重要措施,其中补体C3d分子佐剂倍受人们的关注。
补体C3d分子是补体C3被抗原激活以后的最终裂解片段,能够促进抗原提呈细胞对抗原的摄取、提呈作用,增强机体的免疫应答能力。因此,C3d已经成为核酸疫苗有效的新型分子佐剂。但是C3d作为分子佐剂具有种属差异性,不同种动物间C3d免疫增强作用的差异性需要作进一步的研究。
PRRSV GP5基因编码的GP5蛋白为糖基化囊膜蛋白,该蛋白具有较好的免疫原性,能诱导机体产生特异性体液免疫和细胞免疫。invH基因是沙门氏菌A-E群的高度保守基因,编码吸附和侵袭上皮细胞表面的蛋白,该蛋白决定沙门菌对肠粘膜细胞的侵袭力。
本研究首先对哺乳动物猪、鼠的C3d基因进行克隆及序列测定分析,并探讨了不同种动物(猪、鼠、鸡、鸭)间补体C3d在基因水平上的相关性和差异性,然后以PRRSV GP5为模型基因,利用鼠、猪(哺乳动物)C3d的受体结合功能区p28和鸡、鸭(禽类动物)C3d的受体结合功能区p29作为分子佐剂,构建了C3d-p28(29).n(n=2、4、6)多聚体分子佐剂PRRSV GP5核酸疫苗和沙门氏菌pcDNA3.1-invH-mC3d -p28.6核酸疫苗;用构建的核酸疫苗免疫小鼠并对其免疫效果进行了体液和细胞免疫主要指标的检测,探讨不同动物C3d-p28(29)对核酸疫苗的免疫增强作用。本研究主要包括四部分内容:
1、哺乳动物猪、鼠补体C3d基因的克隆及序列分析:为了获得哺乳动物(猪、鼠)的补体C3d基因克隆并比较同禽类动物(鸡、鸭)C3d基因序列的差异性,首先从哺乳动物鼠、猪的肝组织中提取总RNA,通过RT-PCR扩增C3d基因的cDNA,琼脂糖凝胶电泳鉴定后直接克隆到pMD18-T载体,构建pMD18-T-C3d重组质粒,转化大肠杆菌,酶切鉴定并进行序列测定,然后进行C3d序列和CR2结合区同源性比较分析。电泳结果显示,分别在936bp和888bp处呈现明亮的条带,成功获得了鼠和猪的C3d基因克隆。序列分析结果表明,哺乳动物(猪、鼠)和禽类(鸡、鸭)的补体C3d基因同源性仅为64%;进化树显示,哺乳动物与禽类的亲缘关系越近,C3d基因进化关系也越近。哺乳动物(猪、鼠)与禽类(鸡、鸭)C3d基因的CR2结合区比较分析发现,禽类为29个氨基酸,而哺乳动物为28个氨基酸,两类动物该区氨基酸的同源性仅为62%,而鼠、猪两种哺乳动物之间、鸡、鸭两种禽类动物之间的同源性分别达82.8%和84%,证明补体C3d及CR2结合区具有种属的差异性。
2、不同动物C3d分子佐剂PRRSV GP5核酸疫苗的构建:为探明不同动物C3d分子佐剂在核酸疫苗中的免疫增强作用,在上述试验克隆了动物C3d cDNA的基础上,设计引物克隆C3d-p28(29)至pUC19载体,利用同裂酶BamHⅠ和BglⅡ构建多聚体C3d-p28(29).n,将其克隆至真核表达载体pcDNA3.1(+)上;然后以RT-PCR扩增的PRRSV GP5基因作为模式基因,定向克隆至真核表达载体pcDNA3.1-C3d-p28(29).n中p28(29).n上游,构建pcDNA3.1-GP5-C3d-p28 (29).n重组质粒。酶切结果显示,电泳后在807、987、1167bp处分别出现了明亮的条带,表明成功构建了含有补体C3d-p28(29)多聚体分子佐剂的PRRSV GP5核酸疫苗(pcDNA3.1-GP5-C3d-p28(29).n)。
3、不同动物C3d分子佐剂对PRRSV GP5核酸疫苗免疫增强效果研究:为了探索不同动物C3d分子佐剂对PRRSV GP5核酸疫苗的免疫增强作用及差异性,在构建了鼠、猪、鸡、鸭四种动物补体C3d-p28(29).n PRRSV GP5核酸疫苗(pcDNA3.1-GP5-C3d-p28(29).2.4.6)及pcDNA3.1-GP5核酸疫苗的基础上,分别提取质粒,通过脂质体转染至Marc145细胞进行瞬时表达,并免疫BALB/c小鼠,然后利用不同ELISA试剂盒分别检测各免疫组小鼠的GP5抗体水平和IL-4、IFN-γ含量。结果表明,以补体C3d-p28(29).n为分子佐剂的PRRSV GP5基因重组疫苗均可在Marc145细胞内进行表达;连接不同动物C3d-p28(29)2,4,6聚体的核酸疫苗免疫鼠血清中GP5抗体水平、IL-4和IFN-γ含量均比空载体(pcDNA3.1)和pcDNA3.1-GP5对照组的升高,差异均显著(P<0.05),其中以pcDNA3.1-GP5-p28(29).6组的效果最佳,但均不如PRRSV油乳剂灭活苗组的效果好。另外,含有C3d-p28(29).n(n=2,4,6)相同聚体的疫苗免疫组小鼠血清中的GP5抗体水平、IL-4和IFN-γ含量两两比较无差异性。
4、鼠C3d分子佐剂沙门氏菌invH基因核酸疫苗的构建及免疫效果研究:为了进一步探讨分子佐剂C3d在细菌核酸疫苗中的免疫增强作用,以沙门氏菌invH为核酸疫苗的模式基因,构建了pcDNA3.1-invH-mC3d-p28.6重组质粒,免疫BALB/c小鼠并检测了小鼠血清中invH抗体和IL-4、IFN-γ的含量,然后进行小鼠攻毒保护试验。结果表明,小鼠血清中invH抗体水平、IL-4和IFN-γ的含量与pcDNA3.1、pcDNA3.1-invH对照组比较,差异均显著(P<0.05)。通过攻毒试验结果表明,pcDNA3.1-invH-mC3d-p28.6核酸疫苗对小鼠的免疫保护率高于pcDNA3.1-invH组,差异显著(P<0.05),与空白组和pcDNA3.1组比较,差异均极显著(P<0.01),但核酸疫苗的保护效果不及灭活疫苗。
本研究结果探明了动物C3d分子佐剂对病毒及细菌核酸疫苗的免疫增强作用,为开发利用不同动物C3d分子佐剂研制其他病原的核酸疫苗提供了理论依据和技术支持。
In recent years, with the development of molecular biology and immunology theory and technology, the research of biological products using to prevent and treat have already become a hot spot in the immunology field by genetic restructuring express technology. Among them the nucleic acid (DNA) vaccine is extremely popular. The nucleic acid vaccine is a new generation of vaccine after the inactivated vaccine, live attenuated vaccine, unit vaccine and genetic engineering vaccine, which has many advantages, such as inducing the humoral immune and cellular immune response, safety in the use, easy to produce, and so on. The gene combination vaccine could be made by integrating some genes together. However, the speed of DNA vaccines’application development is limited because its’low protein expression and weaker immune response. Thus, improving the immunity effect of DNA vaccine is an urgent problem to solve in the immune study. At present, application of molecular adjuvants is an important measure to improve the immunity effect of DNA vaccines. The molecular adjuvants have interleukin, interferon, thymosin and complement molecular adjuvants etc, which complement molecules C3d as a new molecular adjuvants is of growing concern.
C3d is the final cleavage product of complement C3 while C3 is activated, which can promote antigen presenting cells (APC) to carry on the uptake of antigens in , to present antigens, and improve the immune response capability. Therefore, C3d has been defined to be a new and effective molecular adjuvant in the nucleic acid vaccine. Whereas C3d as molecular adjuvants has different animal species diversity, the immuno-enhancing differences of C3d among different animals need further study.
GP5 protein is a glycosyl capsule membrane protein coded by PRRSV GP5 gene, which has good immunogenicity and could induce the specific humoral immune and cellular immune. InvH gene is the highly conservative gene at salmonella A-E groups, which can code the surface protein to adsorb and invade epithelial cells. The capacity of salmonella bacteria to invading intestinal mucosa cells is decided by this protein.
Firstly, C3d genes of mammals (pig and mouse) were cloned and sequence analyses were investigated in this study; Subsequently,the relevance and difference between mammals and poultry animals (pig, mouse, chicken and duck) was investigated in gene level. And then, nucleic acid vaccines containing PRRSV GP5 model gene with the complement p28 polymersomes of mammals (swine or mouse) (complement C3d receptor binding domain), or the complement p29 polymersomes of poultry (chicken or duck) and nucleic acid vaccine containing Salmonella invH model gene with C3d-p28.6 of mouse were constructed; mice were inoculated by the nucleic acid vaccines and the main immune indexes of humoral immune and cellular immune were evaluated to observe the immuno-enhancing effect of the different animals C3d (C3d-p28(29).n(n=2、4、6))in the nucleic acid vaccines. This research mainly including four sections:
1. Cloning and Sequence Analysis of Complement Component C3d from the mammals pig and mouse: In order to obtain mammals (pig and mouse) C3d gene cloning and compare the differences with poultry animals (chicken and duck) C3d gene sequences, total cell RNA was extracted from the liver tissue of the mammals (pig and mouse), and the cDNA of C3d was amplified by reverse transcription polymerase chain reaction (RT-PCR). The cDNA fragments were directly cloned into pMD18-T plasmid, and the recombinant plasmids pMD18-T-C3d were identified by restriction endonucleases digestion and sequencing. The cloned C3d genes and CR2 binding region on C3d were compared between mammals and poultry. Electrophoresis showed the C3d gene cloning of pig and mouse were obtained successfully for the bright strips in 936bp and 888bp respectively. The results of sequence analysis indicated that the homology of complement C3d gene between mammals (pig and mouse) and poultry (chicken and duck) is only 64%; the phylogenetic tree showed C3d had varieties in species mammals and poultry, more close relationship, more close evolution. Structural analysis in CR2 binding region indicated that there were 28 amino acids in mammals but 29 amino acids in poultry; the homologous amino acids were only 60% between poultry and mammals, however there were more homologous between mammals pig and mouse 82.8% and between poultry chicken and duck 84%, which indicated that the complement C3d and the CR2 binding region had the genus-specificity.
2. Construction of nucleic acid vaccines containing PRRSV GP5 gene with complement C3d of different animals as molecular adjuvants: In order to find out the immuno-enhancing effect of molecular adjuvant C3d of different animals in the nucleic acid vaccine, after cloning the C3d cDNA, four pairs of primers were designed to subclone the C3d-p28(29) gene to the pUC19 plasmid. Several tandems of C3d-p28(29) were constructed in the pUC19 plasmid used a pair of isoschizomers BamHI and BglII. Digested the pUC19-C3d-p28(29).n to get the gene of C3d-p28(29).n, and then cloned the products to pcDNA3.1 (+) plasmid. After this, the GP5 gene of PRRSV was cloned through RT-PCR and inserted to the upstream of the C3d-p28(29).n which is in the pcDNA3.1-C3d-p28(29).n, and the nucleic acid vaccines containing PRRSV GP5 gene with C3d-p28(29).n as molecular adjuvants were constructed. Electrophoresis showed that the bright strips appear in 807、987、1167bp after digested the reconstructive plasmids (pcDNA3.1-GP5-C3d- p28(29).n) respectively, which indicates that the reconstructive plasmids containing PRRSV GP5 gene with C3d-p28(29).n as molecular adjuvant were constructed successfully.
3. Study on immunity enhancement effect of nucleic acid vaccine containing PRRSV GP5 with C3d of different animal as molecules adjuvants: In order to explore the immuno-enhancing effect and difference of the PRRSVGP5 nucleic acid vaccine containing molecules adjuvant C3d of different animals, the reconstructive plasmids were extracted and expressed instantaneously in the Marc145 cells by liposomes carrying after the reconstructive plasmids of four kinds of animals (pcDNA3.1-GP5-C3d-p28(29).n) and the plasmid (pcDNA3.1-GP5) were constructed. Subsequently, the vaccines’abilities to elicit the humoral and cellular immune responses were investigated in BALB/c mice. The result showed that the reconstructive plasmids well expressed as GP5 protein in the Marc145 cells and that significantly enhanced GP5-specific ELISA antibody, GP5-specific neutralizing antibody, IFN-γlevel, and IL-4 level, could be induced in mice immunized with nucleic acid vaccines encoding the pcDNA3.1-C3d-p28(29).n-GP5 than those received nucleic acid vaccine expressing only the pcDNA3.1 vector and pcDNA3.1- GP5 group (P <0.05), although these were not as effective as inactivated oil-emulsion vaccine. The increase in the immune response elicited by six copies of p28(29) was higher than four copies of p28(29) (P <0.05), which is also higher than two copies of p28(29) (P <0.01). Furthermore, there were no differences of the GP5 antibody level, IL-4 and IFN-gamma content in the serums of mice immunized with nucleic acid vaccines containing the same copies of C3d-p28(29).
4. Construction and immunogenicity of nucleic acid vaccines containing invH gene of salmonella with murine complement C3d as molecules adjuvants: In order to further explore the immuno-enhancing effect of C3d as molecular adjuv- ants in the bacteria nucleic acid vaccine. Nucleic acid vaccines containing the invH as model gene with six copies of mC3d-p28 as molecules adjuvant were constructed; subsequently, mice were inoculated by the nucleic acid vaccines and the invH antibody level, IL-4 and IFN-gamma content in the serums were detected; and then, tapping poison protection test was performed in mice. Result showed differences of the invH antibody level, IL-4 and IFN-gamma content in the serums were significant compared with those received nucleic acid vaccine expressing only the pcDNA3.1 vector and pcDNA3.1-GP5 group (P<0.05).The results of poisoning experiment showed that the protective rate of the vaccine containing six copies of mC3d-p28 was higher than pcDNA3.1-invH group, significant difference (P<0.05), and was extremely significant difference (P<0.01)compared the blank group and pcDNA3.1 group, although these were not as effective as inactivated oil-emulsion vaccine.
The results of this study has proven the immuno-enhancing effect of molecular adjuvants C3d in the viruses and bacteria nucleic acid vaccine, which may provide theoretical basis and technical support for the development of other pathogens’nucleic acid vaccine using C3d of different animals as molecular adjuvants.
引文
程之范.20世纪后半期的疫苗[J].中华医史杂志,1997,27(3):185-190
褚新星,彭军,翁立雪等.新城疫C3d-P29分子佐剂F基因疫苗的构建.微生物学报, 2008,48(2):234-238
崔保安,魏战勇,王学斌等. IL-2与猪细小病毒VP_2基因双表达载体的构建及免疫原性的研究.生物工程学报,2006,22(3):425-429
崔保安,刘占通,文英会等.猪繁殖与呼吸综合征病毒分子生物学研究进展[J].动物医学进展,2004,25(1):22-24
杜德伟,周永兴,冯志华,等.IL-12的真核表达载体促进小鼠对DNA疫苗的免疫应答[J].中华微生物学核免疫学杂志,2000,20(6):243-544
方新华. DNA疫苗的结构与免疫应答的关系.中华医学杂志,2001,25(3):159-160
方艳秋,许淑芬,谭岩. TNF-α,IL-6,IL-4,IL-2对IFN-γ抗弓形虫感染作用的影响[J].免疫学杂志,2000,16(3):196-199
龚伟,金宁一,薛立娟等. NDV长春株和四平株HN/F核酸疫苗的构建及表达.中国兽医学报,2002,22(2):105-107
关庆东,王立新,徐薇等.HBV-pre-2/S-C3d联合基因疫苗诱导的特异性免疫应答及其意义.中国免疫学杂志,2006,22(9):813-821
郭海龙.猪繁殖与呼吸综合征病毒感染猪体内细胞因子与病毒的定量检测[D].北京:中国农业科学院,2002
黄秀琴,陈冬梅,倪晓华.不依赖特异性抗体激活补体经典途径的机理探讨[J].华东师范大学学报(自然科学版), 1995, 3:90-93
黄秀琴,唐鹰,吴晶琼等.大肠杆菌不依赖特异性抗体激活补体系统经典途径的研究[J].华东师范大学学报(自然科学版),1994,2:98-101
江云波,方六荣,肖少波,等.猪繁殖与呼吸综合征病毒GP5和M蛋白共表达的自杀性DNA疫苗的构建及其免疫应答[J].中国农业科学, 2006, 39: 1011-1017
黎川,朱鸿飞.羊补体分子C3d基因的克隆及羊包虫病基因工程亚单位疫苗的研制[D]。中国农业科学院硕士论文, 2008
李海山,刘勇.DNA疫苗及其优化策略[J].国外医学(病毒学分册),2003,10 (3):74-78
李建伟,赵晓岩,杨建德等.鸡马立克氏病DNA疫苗免疫的初步研究.中国畜禽传染病,1996,5:9-12
李忠明.当代新疫苗.北京:高等教育出版社,2001
刘光清,薛强,仇华吉,等.猪繁殖与呼吸综合征病毒CH-1a株非结构基因的分子克隆及其基因特征的研究[J].中国预防兽医学报,2002,24(2):81-87
罗琴芳,金宁一,钱剑.鸡新城疫病毒(NDV)HN蛋白基因核酸疫苗的构建与血凝活性测定.塔里木农垦大学学报,2002,14(2):1-5
孟松树,张绍杰,童光志等.鸡传染性喉气管炎病毒ONA疫苗免疫效果观察.中国预防兽医学报,2000,22(3):179-151
PRRSV感染的仔猪体内病毒抗原分布和临床病理变化研究[D].新疆乌鲁木齐.新疆农业大学硕士论文, 2002
舒俭德.人用疫苗:回顾与展望[Jl.中国计划免疫,2000,6(2):117-121
孙树汉.核酸疫苗.上海:第二军医大学出版社,2000:149-152
田明礼,易新元,曾宪芳,等.日本血吸虫31 Kda组织蛋白酶B DNA疫苗保护性免疫效果观察[[J].中国血吸虫病防治杂志,2001,13(4):209-212
童光志,仇华吉,周彦君,等.猪生殖-呼吸道综合征病毒CH-1a株结构蛋白基因的克隆与序列分析[J].自然科学进展,2000,10(2):147-153
王立新,关庆东,徐薇等.C3d-P28对基因免疫诱导的HBV特异性细胞免疫应答的调节作用.中国免疫学杂志,2004,20(7):443-447
王立新,徐薇,关庆东.C3d-p28增强乙型肝炎病毒特异性基因免疫效果的研究.中华微生物学和免疫学杂志,2003,23(6):423-427
王秀丽,李大金,袁敏敏等.分子佐剂C3d3增强hCGβ蛋白疫苗免疫原性及其抗血清中和hCGβ生物学活性的作用.实验生物学报,2004,37:255-261
王章云.肠炎沙门氏菌引起的食物中毒细菌学调查[J].中国人畜共患病杂志,1999,15(3):115
魏泉德,叶荃,徐劲,等.DNA疫苗在小鼠体内组织分布及安全性研究[[J].中国人兽共患病杂志,2001,17(5):9-17
吴乃虎编著.基因工程原理[[J].北京:科学出版社,2001:474-481
殷震,刘景华主编.动物病毒学.北京:科学出版社,1997,619-625
余敏,李大金,王秀丽等.hCGβ-C3d3基因免疫BALB/c小鼠选择性促进B细胞克隆扩增.现代免疫学,2006,26(1):17-20
余敏,李大金,王秀丽等.分子佐剂C3d上调Raji细胞协同刺激分子B7-1和B7-2的表达.分子生物学报,2006,39(1):78-81
章振华,周雪媚,谌南辉.鸡补体基因C3d的基因克隆及结构分析.生物技术通讯,2006,17:567-570
赵欣荣,李大金,蔡立荣等.分子佐剂C3d增强hCGβ基因免疫体液免疫效应.中国免疫学杂志,2003,19(9):619-624
赵修竹.补体系统的有限蛋白解[J].上海免疫学杂志,1997,17(4):193-196 赵修竹.补体学.湖北科学技术出版社,长沙,1998
郑春福.DNA疫苗的载体及其主要结构特点[J].国外医学(生物制品分册),2000, 23(4):146-150
郑春福,吴少庭,陈雅棠,等.恶性疟原虫DNA疫苗在小鼠体内组织分布和安全性初步研究[[J].中国人兽共患病杂志,2002,18(6):29-31
邹强,郑萍,等.小鼠补体C3分子cDNA的克隆和鉴定[J].免疫学杂志,2003,3:886-890
Akbari O.,Panjwani N.,Garcia S. DNA vaccination: transfection and activation of dendritic cells as key events for immunity. J ExP Med, 1999, 189(1): 169-178
Allende R., Lewis T. L., Lu Z., et al. North American and European porcine reproduc- tive and respiratory syndrome virus differ in non-structural protein coding regions [J]. J Gen Virol, 1999, 80(2):307-315
Andrea E. P., David R., Thilo S., et al. The crystal structure of human CD21: implications for Epstein barr virus and C3d binding [J]. Proc Natl Acad Sei USA, 2002, 99 (16): 10 641-10646
Ansari I..H., Kwon B., Osorio F. A., et al. Influence of N-linked glycosylation of porcine reproductive and respiratory syndrome virus GP5 on virus infectivity, antigenicity, and ability to induce neutralizing antibodies [J]. American Society for Micro- biology, 2006, 3994-4004
Anu Cherukuri, Paul C., Cheng, Susan K., et al. The role of the CD19/CD21 complex in B cell processing and presentation of complement-tagged antigens[J]. The Journal of Immunology, 2001, 167: 163-172
Astorza de B., Cortes G., Crespi C., et al. C3 promotes clearance of Klebsiella pneumoniae by A549 epithelial cells [J]. Infect Immun, 2004, 72(3): 1767-1774
Atkinson C., Song H., Lu B., et al. Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent increase in susceptibility to infection. J Clin Invest, 2005, 115(9): 2444-2453
Aydar Y.,Sukumar S.,Szakal A. K.,Tew J. G.. The influence of immune complex-bearing follicular dendritic cells on the IgM response,Ig class switching and production of high affinity IgG. J Immunol,2005, 174: 5358–5366
Barel M., Balbo M., Romancer M., Frade R.. Activation of Epstein- Barr virus/C3d receptor (gp140, CR2, CD21) on human cell surface triggers pp60src and Akt-GSK3 activities upstream and downstream to PI3-kinase, respectively. Eur J Immunol, 2003: 33(9): 2557-2566
Barfoed A. M., Blixenkrone M. M., Jensen M. H., et al. DNA vaccination of pigs with open reading frame 1-7 of PRRS virus [J].Vaccine, 2004, 22(27-28):3628-3641
Barry M. A., Johnston S. A.. Biological features of genetic immunization. Vaccine, 1997, 15(8): 788-791
Batista L., Pijoan C., Dee S., et al. Virological and immunological responses to porcine reproductive and respiratory syndrome virus in a large population of gilts [J] . Can J Vet Res, 2004, 68(4): 267-273
Batista L., Pijoan C., Dee S., et al. Virological and immunological responses to porcine reproductive and respiratory syndrome virus in a large population of gilts [J].Can J Vet Res, 2004, 68(4): 267-273
Baustista E. M., Meulenberg J. J., Choi C. S., et al . Structural polypeptides of the American (VR-332) strain of porcine epidemic abortion and respiratory virus [J]. Arch Virol, 1996, 141: 1357-1365
Bautista E. M., Goyal S. M., Meulenberg J. J., et al. Structural polypeptides of the American (VR-2332) strain of porcine reproductive and respiratory syndrome virus[J]. Arch Virol, 1996, 141:1357-1365
Bautista E.M., Molitor T.W.. IFN gamma inhibits porcine reproductive and respiratory syndrome virus replication in macrophages [J]. ArchVirol, 1999, 144 (6):1191– 1200.
Baxrington R., Zhang M., Fischer M., et al. The role of complement in inflamrnation and adaptive imrnunity[J]. Imrnunol Rev, 2001, 180: 5
Benfield D. A., Nelson E., Collins J. E., et al. Characterization of swine infertility and respiratory syndrome(SIRS)virus(isolate ATCC VR-2332)[J]. J Vet Diagn Invest, 1992, 4:127-133
Bergmann-Leitner E. S., Duncan E. H., Leitner W. W., et al. C3d-defined complement receptor-binding peptide p28 conjugated to circumsporozoite protein provides protection against Plasmodium berghei. Vaccine, 2007, 25(45):7732-7736
Bethesda M. D.. Workshop on the control and standardization of nucleic acid vaccines. Vaccine, 1994, 12(16): 1526-1528
Bethesda M.D.. Workshop on the control and standardization of nucleic acid vaccines.Vaccine, 1997, 15: 931-933
Bower J. F., Green T. D., Ross T. M.. DNA vaccine expressing soluble CD4 envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV1. Virology, 2004, 328(2): 292-300
Bower J. F., Sanders K. I., Ross T. M.. C3d enhances immune responses using low doses of DNA expressing the HIV-1 envelope from codon-optimized gene sequences. Curr HIV Res, 2005, 3(2): 191-198
Cancel-Tirado S. M., Evans R. B., Yoon K. J., et al. Monoclonal antibody analysis of porcine reproductive and respiratory syndrome virus epitopes associated with antibody-dependent and neutralization of virus infection [J]. Vet Immunol Immunopathol. 2004, 102(3):249-262
Chen S. C., Fynan E. F., Greenberg H. B., et al. 1999. Immunity obtained by gene-gun inoculation of a rotavirus DNA vaccine to the abdominal epidermis of anorectal epithelium.Vaccine. 17:3171-3176
Cheng A. C., Wang M. S., Chen X. W.. Cellular immune responses of BALB/ c mice induced by intramuscular injection of PRRSV ORF5 DNA vaccine with different doses [J]. Agric, 2007, 1(1): 105-110
Cherukuri A., Cheng P. C., Pierce S. K.. The role of the CD19/CD21 complex in B cell Processing and Presentation of complement-tagged antigens. J Immunol. 2001, 167(1): 163-172
Cho J. G.., Dee S. A.. Porcine epidemic abortion and respiratory syndrome virus[J].Theriogenology, 2006, 66:655-662
Clemenza L., Isenman D.E.. Structure-guided identification of C3d residues essential for its binding to complement receptor 2 (CD21). J Immunol, 2000, 165(7): 3839-48
Colomb M.G., Bensa J.C., Chesne S., Aubert B.. Interactions between complement system and bacterial walls[J]. Bull Eur Physiopathol Respir, 1983, 19(2): 131-136
Cooper V. L., Doster A. R., Hesse R. A.. Porcine reproductive and respiratory syndrome: Neb-1 PRRSV infection did not potentiate bacterial pathogens [J]. J Vet Diagn Invest, 1995, 7(3):313-320
Creasey E. A., Friedberg D., Shaw R. K., et al. CesAB is an enteropathogenic Escherichia coli chaperone for the type-Ⅲtranslocator proteins EspA and EspB. Microbiology, 2003, 149: 3639-3647
Davis H. L., Michel M. L., Macini M.. Direct gene transfer into skeletal muscle: plasmid DNA-based immunization against the hepatitis B virus surface antigen [J].Vaccine,1994,12: 1503-1509
Davis H L., Whalen R. G., Demeneix B. A.. Direct gene transfer into skeletal muscle in vivo: Factors affecting efficiency of transfer and stability of expression. Human Gene Therapy, 1993, 4(2): 151-159
Dea S., Wilson L., Therrien D.. Competitive ELISA for detection of antibodies to porcine reproductive and respiratory syndrome virus using recombinant E.coli-expressed nucleocapsid protein as antigen[J]. J Virol, 2000, 87:109-122
Dea S., Gagnon C.A., Mardassi H., et al. Current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (PRRS) virus: comparison of the North American and European isolates. Arch Virol, 2000, 145: 659-688
Dee S.A., Joo H., Park B.K.. Attempted elimination of porcine reproductive and respiratory syndrome virus from a seedstock farm by vaccination of the breeding herd and nursery depopulation [J]. Vet Rec, 1998, 142(21):569-57.
Dela Cruz C. S., MacDonald K. S., Barber B. H..Anti-major histocompatibility complex antibody responses in macaques via intradermal DNA immunizations[J]. Vaccine, 2000, 18(27): 3152-3165
Deliis G., Boyle J. S., Brady J. L.,et al. A fusion DNA vaccine that targets antigen-presentingcells increases protection from viral challenge [J]. Proc Acad Sci, 2000, 6676-6680
Dempsey P. W., Allison M. E., Akkaraju S., et al. C3d of complement as a molecular adjuvant: Bridging innate and acquired immunity. Science, 1996, 271(5247): 345-350
Dempsey P. W., Allison M. E., Akkaraju S., et al. C3d of complement as a molecular adjuvant: Bridging innate and acquired immunity. Science, 1996, 271(5247): 345-350
Denac H., Moser C., Tratschin J. D., et al. An indirect ELISA for the detection of antibodies against porcine reproductive and respiratory syndrome virus using recombinant nucleoapsid protein as antigen [J].J Virol,1997, 65:169-181
Deng W., Puente J. L., Gruenheid S., et al. Dissecting virulence: Systematic and functional analyses of apathogenicity island. Proc Natl Acad Sci USA, 2004, 101: 3597-3602
Dimitrios M., John D. L..The electrostatic nature of C3d complement receptor 2 association [J]. J Immunol, 2004, 172:7537-7547
Donald L., Nancy B.,Ewalt LC. Gene gun partical-mediated vaccination with plasmid DNA confers protective immunity against rabits virus infection[J].Vaccine, 1998 16(2/3): 115-118
Donnelly J. J., Ulmer J. B., Shiver J. W., et al. DNA vaccines. Annu Rev Immunol, 1997, 15: 617-648
Drew T. W.. A review of evidence for immuno-suppreesion due to porcine reproductive and respiratory syndrome virus (PRRSV)[J].Vet Res, 2000, 31(1):27-39
Elliott S. J., Koutsouris A., Kaper J. B., et al. A gene from the locus of enterocyte effacement that is required for enteropathogenic Escherichia Coli to increase tight-junction permeability encodes a chaperone for EspF. Infect Immun, 2002, 70:2271-2277
Esparza I., Becherer J. D., Alsenz J., et al. Evidence for multiple sites of interaction in C3 for complement receptor type 2 (C3d/EBV receptor, CD21). Eur J Immunol, 1991, 21: 2829-2838
Faaberg K.S., Hocker J.D., Erdman M.M., et al. Neutralizing Antibody Responses of Pigs Infected with Natural GP5 N-Glycan Mutants of Porcine reproductive and Respiratory Syndrome Virus[J].Viral Immunol, 2006, 19:294-304
Fang L. R., Jiang Y. B., Xiao S. B., et al. Enhanced immunogenicity of the modified GP5 ofporcine reproductive and respiratory syndrome virus [J]. Virus Genes, 2006, 32: 5-11
Fearon D. T., Carroll M. C.. Regulation of B lymphocyte responses to foreign and self antigens by the CD21/CD19 complex. Ann Rev Immunol, 2000, 18: 393-422
Fernandez A., Suarez P., Cast ro J. M., et al. Characterization of regions in the GP5 protein of porcine reproductive and respiratory syndrome virus required to induce apoptotic cell death [J].Virus Res, 2002, 83(1-2): 103-118
Fischer M. B., Goerg S., Shen L.M., et al. Dependence of germinal center B cells on expression of CD21/ CD35 for survival [J]. Science, 1998, 280(5363): 582 - 585
Fodior I., Horvath E., Fodor N., et al. Induction of Protective immunity in chickens immunized with Plasmid DNA encoding infectious bursa disease virus antigens. Acta Vet Hung, 1999, 47(4): 481~492
Forshtuber T., Yip H. C., Lehmann P. V.. Induction of TH1 and TH2 immunity in neonatal mice[J]. Science, 1996, 271(5256): 1728-1730
Fu T..M., Ulmer J. B., Caulfield M. J., et al. 1997. Priming of cytotoxic T lymphocytes by DNA vaccines: requirement for professional antigen presenting cells and evidence for antigen transfer from myocytes. Mol Med. 3(6):362-71
Fynan E.F., Webster R.G.., Fuller D.H., et al. 1993. DNA vaccines: Protective immunizations by parenteral, mucosal, and gene-gun inoculations. Proc. Natl. Acad. Sci. USA. 90:11478-11482
Fynan E. F., Robinson H. L., Webster R. G.. Use of DNA encoding influenza hemagglu- tinin as an avian influenza vaccine DNA[J]. Cell Biol, 1993, 12(9):785-789
Fynan E. F.,Webster R. G., Fuller D. H., et al. DNA Vaccine: protective immunizations by parenteral: muscocal and gene-gun inoculations [J].Proc Natl Acid, 1993 , 90 (24): 11478-11482
Galina L., Pijoan C., Sitjar M., et al. Interaction between Streptococcus suit serotype 2 and porcine reproductive and respiratory syndrome virus in specific pathogen- free piglets [J].Vet Rec, 1994, 134(3):60-64
Gerda S., Guthridge J. M., Li D. W., et al. Structure of complement receptor 2 in complex with its C3d ligand[J]. Science, 2001, 292: 1725-1728
Gilbert H. E., Eaton J. T., Hannan J. P., et al. Solution structure of the complex between CR2 SCR 1-2 and C3d of human complement: an X-ray scattering and sedimentation modeling study. J Mol Biol, 2005, 346(3): 859-873
Gilbert H. E., Hannan J., Holers V. M., et al. Synchrotron X-ray scattering and ultracentri- fugation of unbound CR2 SCR1-2 shows that SCR-1and SCR- 2 form a family of extended V-shapes in solution. Mol.Immunol, 2004: 41: 235
Gonin P., Mardassi H., Gagnon C.A., et al. A nonstructural and antigenic glycoprotein is encoded by ORF3 of the IAF-Klop strain of porcine reproductive and respiratory syndrome virus[J].Arch Virol, 1998, 143:1927-1940
Grebennikova T.V., Clouser D.F., Vorwald A.C., et al. Genomic characterization of virulent,attenuated and revertant passages of a North American Porcine reproductive and respiratory syndrome viruse strain[J].Virology,2004, 321:383- 390
Green D. T., Newton B. R., Rota R. A., et al.C3d enhancement of neutralizing antibodies to measles’hemagglutinin. Vaccine, 2002, 20:242-248
Green T. D., Montefiori D. C., Ross T. M.. Enhancemant of antibodies to the human immunodeficiency virus type 1 envelope by using the molecular adjuvant C3d. J Virol, 2003, 77(3):2046-2055
Greenland J. R., Letvin N. L..Chemical adjuvants for plasmid DNA vaccines.Vaccine, 2007, 25(19):3731-3741
Gregoriadis G.. Genetic vaccines: Strategies for optimization. Pharmaceutical Research, 1998, 15(5): 661-670
Guthridge J. M., Young K., Gipson M. G., et al. Epitope mapping using the X-ray crystallographic structure of complement receptor type 2 (CR2)/CD21: identification of a highly inhibitory monoclonal antibody that directly recognizes the CR2-C3dinterface. J Immunol, 2001, 167(10): 5758-5766
Haas K. M., Hasegawa M., Steeber D. A., et al Complement receptors CD21/35 link innate and protective immunity during Streptococcus pneumoniae infection by regulating IgG3 antibody responses. Immunity, 2002, 17(6): 713-723
Haddad D., Liljeqvist S.. Comparative study of DNA -based immunization vectors: effect of secretion signals on the antibody responses in mice. FEMS Immunol Med Microbiol,1997, 18: 193-202
Halbur P. G.., Paul P. S., Frey M. L., et al. Comparison of the pathogenicity of two U.S. porcine reproductive and respiratory syndrome virus isolates with that od Lelystad virus [J]. Vet Pathol, 1995, 32:648-660
Hannah E. G., Julian T. E., Jonathan P. H., et al. Solution Structure of the Complex between CR2 SCR 1-2 and C3d of Human Complement: An X-ray Scattering and Sedimentation Modeling Study. J. Mol. Biol, 2005, 346, 859–873
Hannan J., Young K., Szakonyi G., et al. Structure of complement receptor (CR) 2 and CR2-C3d complexes. Biochem Soc Trans, 2002, 30(Pt 6): 983-989
Hannan J. P., Young K. A., Gutblidge J. M., et al. Mutational analysis of the complement receptor type 2 (CR2/CD21)-C3d interaction reveals a Putative charged SCR1 binding site for C3d. J Mol Biol, 2005, 346 (3): 845-58
Hariet L., Robinson. Nucleic acid vaccines an overview.Vaccine, 1997, 15: 785-787 Hauser H., Chen S. Y.. Augmentation of DNA Vaccine Potency through secretory heat shock Protein-mediated antigen targeting. Methods, 2003, 31(3): 225-231
Heather I., Davis H. L., Robert G., et al. Direct gene transfer into skeletal muscles in vivo: factors alerting efficiency of transfer and stability of expression [J].Hun Gene Then 1993,4: 151-159
Hess M. W., Schwendinger M. G., Eskelinen E. L., et al. Tracing uptake of C3dg-conjugated antigen into B cells via complement receptor type 2 (CR2, CD21).J Blood, 2000, 95: 2617–2623
Ihata A.,Watabe S.,Sasaki S.,et al .Immunomodulatory effect of a Plasmid expressing CD40 ligand on DNA vaccination against human immunodeficity virus type-1[J]. Immunology, 1999 98(3): 436-442
Isabelle Nobiron, Ian Thompson, Joe Brownlie, et al. Cytokine adjuvancy of BVDV DNA vaccine enhances both humoral and cellular immune responses in mice [J]. Vaccine, 2001, 19(30): 4226-423
Iwasaki A., Torres C. A., Ohashi P. S., et al. The dominant role of bone marrow-derived cells in CTL induction following plasmid DNA immunization at different sites. J Immunol, 1997, 159(1):11-4
Jiang Y. B., Fang L. R., Xiao S. B., et al. Construction and immunogenicity of recombin- ant pseudorabies virus expressing the modified GP5 protein of porcine reproduction and respiratory syndrome virus [J]. Biol, 2007, 2(1): 85-91
Johnson C.R., Yu W.Q., Murtaugh M.P.. Cross-reactive antibody responses to Nsp1 and Nsp2 of porcine reproductive and respiratory syndrome virus[J].J Gen Virol, 2007, 88:1184-1195
Jon Cohen. Naked DNA points way to vaccines. Science, 1993, 259:1691
Jonathan P. H., Kendra A. Y., Joel M. G., et al. Mutational analysis of the complement receptor type 2(CR2/CD21)C3d interaction reveals‘a putative charged SCR1 binding site for C3d [J].J Mol Biol, 2005,346: 845-858
Kim T. W., Hung C. F., Cheng W. F., et al. Tumor-specific immunity and anti-angiogenesis generated by a DNA Vaccine encoding calreticulin linked to tumor antigen. J Clin Invest, 2001, 108(5): 669
Kingsley R. A., Humphries A. D., Weening E. H., et al. Molecular and phenotypic analysis of the C554 island of Salmonella enterica serotype typhimurium: Identification of intestinal colonization and persistence determinants [J]. Infect Immun, 2003, 71(2):629-640.
Klavinskis L. S., Batnfield C., Gao L., et al. Intranasal immunization with plasmid DNA-lipid complexes elicits mucosal immunity in the female genital and rectal tracts. J Immunol, 1999, 162: 254-262
Koch M., Frazier J., Sodroski J., et al. Characterization of antibody responses to Purified HIV -1 gP120 glycoproteins fused with the molecular adjuvant C3d. Virology, 2005, 340(2): 277-284
Kodihalli,Haynes J. R., et al.Crossprotection among lethal HSN2 influnza viruses induced by DNA vaccine to the hemagglutinin[J].Virol, 1997, 71:3391-3396
Kusakabe K., Xin K. Q., Katoh H., et al. The timing of GM-CSF expression Plasmid administration influences the Thl/Th2 response induced by an HIV-l-specific DNA Vaccine. J Inununol, 2000, 164(6): 3102-3111
Lagging L. M., Meyer K., Hoft D., et al. Immune responses to plasmid DNA encoding the hepatitis C virus core protein. J Virol, 1995,69: 5859-5863
Lambris J. D., Ganu V. S., Hirani S., et al. mapping of the C3d receptor (CR2)-binding siteand a neoantigenic site in the C3d domain of the third component of complement. Proc Natl Acad Sci USA, 1985, 82: 4235-4239
Lee Y, Haas K. M., Gor D. O., et al. Complement component C3d-antigen complexes can either augment or inhibit B lymphocyte activation and humoral immunity in mice depending on the degree of CD21/CD19 complex engagement. J Immunol, 2005, 175(12): 8011-8023
Leifert J. A., Lindencrona J. A., Charo J., et al. Enhancing T cell activation and antiviral protection by introducing the HIV-l protein transduction domain into a DNA vaccine.Hmu Gene Ther, 2001, 12(15): 1881-1892
Leslie R. G., Prodinger W. H., Nielsen C. H.. Complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) cooperate in the binding of hydrolyzed complement factor 3 (C3i) to human B lymphocytes [J]. Eur J Immunol, 2003, 33(12):3311-21
Li D. J., Wang H. M., Lei Li., et al. Gene Fusion of Molecular Adjuvant C3d to hCGβenhances the anti-hCGβAntibody Response in DNA immunization. J Re Prod Immunol, 2003, 60: 129-141
Li H., Yang H.. Infection of porcine reproductive and respiratory syndrome virus suppresses the antibody response to classical swine fever virus vaccination [J]. Vet Microbiol. 2003, 95(4):295-301
Lillehoj H. S., choi K. D., Jenkins M. C., et al. A recombinant Eimeria Protein inducing interferon-gamma Production: comparison of different gene expression systems and immunization strategies for vaccination against coccidiosis. Avian Disease, 2000, 44(2): 379-389
Lima J., Jeknins C.,Guerrero A., et al. A DNA vaccine encoding genetic fusions of carcinoembryonic antigen (CEA) and granulocyte/macrophage colonystimulat- ing factor (GM-CSF). Vaccine, 2005, 23(10): 1273-1283
Linsley P. S.,Breay W., et al. Bingding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation [J].J Exp Med, 1991, 173:721-730
Liu F., Mboudjeka I., Shen S., et al. Independent but not synergistic enhancement to the immunogenieity of DNA Vaccine expressing HIV-l gP120 glycoprotein by codonoptimization and C3d fusion in a mouse model. Vaccine, 2004, 22(13-14): 1764-1772
Liu F. J., Innocent M boudjeka, Lu S., et al. Independent but not synergistic enhancement to the immunogenicity of DNA vaccine expressing HIV-1 gp120 glycoprotein by codonoptimization and C3d fusion in a mouse model. Vaccine, 2004, 22: 1764–1772
Lodmell D.L., Ray N.B., Ewalt L.C.. Gene plasmid DNA confers protective immunity against rabies gun particle-mediated vaccination with virus infection. Vaccine, 1998, 16:115-118.
Loemba H.D., Mounir S., Mardassi H.. Kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus [J].Arch Virol, 1996, 141(3-4):751-761
Lohse L., Nielsen J., Eriksen L.. Temporary CD+8 T-cell depletion in pigs’dose not exacerbate infection with porcine reproductive and respiratory syndrome virus (PRRSV) [J].Viral Immunol, 2004, 17(40):594-603
Lou D., Kohler H.. Enhanced molecular mimicry of CEA using photo affinity crosslinked C3d peptide. Nat Biotechnol, 1998, 16(5): 458-462
Lowrie D. B., Cilva C. L., Colston M. J., et al. Protection against tuberculosis by a plasmid DNA vaccine [J].Vaccine, 1997, 15:834-838
Lyubchenko T., Porto J., Cambier J. C., et al. Colligation of the B cell receptor with complement receptor type 2 (CR2/CD21) using its natural ligand C3dg: activation without engagement of an inhibitory signaling pathway. J Immunol, 2005, 174 (6): 3264-3272
Manoj S., Griebel P. J., Babiuk L. A., et al. Targeting with bovine CD154 enhances humoral immune responses induced by a DNA vaccine in sheep. J Immunol, 2003, 170(2): 989-996
Manoutcharian K., Terrazas L. I., Gevorkian G., et al. Protection against murine cysticercosis using cDNA expression library immunization[J].J Immunol lett,1998,62 (3): 131-137
Marches O., Ledger T. N., Boury M., et al. Enteropathogenic and Enterohaemorrhagic Escherichia coli deliver a novel effector Called Cif, which blocks cell cycleG2/M transition. Mol Microbiol, 2003, 50:1553-1567
Mardassi H., Mounir S., Wilson L., et al. Identification of major differences in the nucleocapsid protein genes of a Quebec strain and European strains of porcinereproductive and respiratory syndrome virus[J].J Gen Virol,1994, 75:681-685
Mardassi H., Mounir S., Dea S.. Molecular analysis of the ORF3-7 of porcine reproductive and respiratory syndrome virus, Quebec reference strain[J].Arch Virol, 1995, 140:1405-1418
Margaret D., Mooree N. R., Cooper B. F., et al. Molecular cloning of the cDNA encoding the Epstein-Barrvirus/C3d receptor (complement receptor type 2) of human B lymphocytes. Proe Nati Aead Sci.1987: 84: 9194-9198
Martin T., Parker S. E., Hedstrom R., et al. Plasmid DNA malaria Vaccine: the potential for genornic integration after intramuscular injection [J].Hun Gen Ther, 1999, 10 (5): 759-761
Masilamani M., Seydlitz E., Bastmeyer M., et al. T cell activation induced by cross-linking CD3 and CD28 leads to silencing of Epstein-Barr virus/C3d receptor (CR2/CD21) gene and protein expression. Immunobiology, 2002, 206(5): 528-536
Maue A. C., Waters W. R., Palmer M. V., et al. CD80 and CD86, but not CD154, augment DNA Vaccine-induced Protection in experimental bovine tuberculosis.Vaccine, 2004, 23(6): 769-779
Mendoza R. B.,Cantwell M. J., et al. Cuting edge: immunostimulatory of a plasmid expressing CD40 ligand (CD154) on gene immunization [J]. J Immunol, 1997, 59 (12): 5777-5781
Meng X. J., Paul P. S., Halbur P. G.., et al. Phylogenetic analyses of the putative M(ORF6) and N(ORF7) genes of porcine reproductive and respiratory syndrome virus(PRRSV): implication for the existence of two genotypes of PRRSV in USA and Europe[J].Arch Virol, 1995, 40:745-755
Meri T., Murgia R., Stefanel P., et al. Regulation of complement activation at the C3 level by serum resistant [J]. Leptospires Microb Pathog, 2005, 39(4): 139-47
Meulenberg J. J. M., Nieuwstadt A. P., Essen Z. A., et al. Posttranslational processing and identification of a neutralization domain of the GP4 protein encoded by ORF4 of Lelystad virus [J].J Virol, 1997, 71:6061-6067
Michael A. B., Stephen A. J..Biological features of genetic immunization [J]. Vaccine, 1997, 15(8): 788-791
Min W., Lillehoj H. S., Burnside J., et al. Adjuvant effects of IL-beta, IL-2, IL-8, IL-15, IFN-alpha, IFN-gamma GF-beta4 and lymphotactin on DNA vaccination against Eimeria acervulina. Vaccine, 2001, 20(l-2): 167-174
Molina H., Perkins S. J., Guthridge J., et al. Characterization of a Complement Receptor 2 (CR2/CD21) ligand binding site for C3. J.Irnpnunol.1995, 154: 5426-5435
Molitor T. W., Bautista E. M., Choi C. S.. Immunity to PRRSV: Double-edged sword [J]. Vet Microbio, 1997, 55(1-4):265-276
Mor G., Singla M., Steinberg A. D., et al. Do DNA vaccines induce autoimmune disease? Hum Gene Ther, 1997, 8:293 - 300
Morikis D., Lambris J.D..The electrostatic nature of C3d-complement receptor 2 asso- ciation. J Immunol, 2004, 172(12): 7537-7547
Morozov I.., Meng X. J., Paul P. S., et al. Sequence analysis of open reading frames(ORFs) 2 to 4 of a US isolate of porcine reproductive and respiratory syndrome virus[J].Arch Virol, 1995, 140:1313-1319
Mueller Ortiz S. L., Drouin S. M., Wetsel R. A.. The alternative activation pathway and complement component C3 are critical for a protective immune response against psedomomas aeruginosa in a murine model of pneumonia [J]. Infct Immun, 2004, 72(5): 2899-2906
Mundy R., Jenkins C., Yu J., et al. Distribution of espamong Clinical enterohaemorrhagic and enteropathogenic Escherichia Coli isolates. Med Microbiol, 2004, 53: 1145-1149
Murtaugh M. P., Xiao Z. G.., Zuckermann F.. Immunological responses of porcine reproduc- tive and respiratory syndrome virus infection[J].Viral Immunol, 2002, 15(4):533-547
Murtaugh M P., Xiao Z., Johnson C. R., et al. Porcine immunity to porcine reproductive and respiratory syndrome virus ( PRRSV): systemic and local response in acute and persistent infection[J]. Proceedings of the IX International Symposium on Nido viruses (Arteri viruses and Corona viruses), 2003, (7):61
Nagar B., Jones R. G.., Diefenbaeh R. J., et al. X-ray crystal structure of C3d: a C3 fragment and ligand for complement receptor 2. Seience, 1998, 280(5367): 1277-1281
Nagar B., Jones R. G.., Diefenbaeh R. J., et al. X-ray crystal structure of C3d: a C3 fragmentand ligand for complement receptor 2. Seience, 1998, 280(5367): 1277-1281
Narita M., Ishii M.. Encephalomalacic lesions in pigs dually infected with porcine reproduc- tive and respiratory syndrome virus and pseudorabies virus [J]. J Comp Pathol, 2004, 131(4):277-284
Nobiron I., ThomPson I., Brownlie J., et al. Cytokine adjuvnacy of BVDV DNA vaccine enchances both humoral and cellular inunune responses in mice. Vaccine, 2001, 19(30): 4226-4235
Ostrowski M., Galeota J. A., Jar A. M., et al. Identification of neutralizating and nonneu- tralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain [J]. J Virol. 2002, 76(9):4241-4250
Parker S. E., Borellini F., Wenk M. L.. Plasmid DNA malaria vaccine: tissue distribution and safety studies in mice and rabbits. Hum Gene Ther, 1999, 10(5): 741-758 Parker S. E., Monteith D., Horton H., et al. Safety of a GM-CSF adjuvant-plasmid DNA malaria vaccine. Gene Ther, 2001, 8(13): 11-23
Pertmer T. M., Eisenbraun M. D., Mc Cabe D., et al. Gene gun-based nucleic acid immunization: Elicitation of humoral and cytotoxic T lymphocyte responses following epidermal delivery of nanogram quantities of DNA. Vaccine, 1995, 13:1427-1430
Pirzadeh B., Dea S.. Monoclonal Antibodies to the ORF5 product of porcine reproductive and respiratory syndrome virus define linear neutralizing determinants. J Gen Virol, 1997, 78(8):1867-1873
Pirzadeh B., Dea S., et al. Immune response in pigs vaccinated with plasmid DNA encoding ORF5 of porcine reproductive and respiratory syndrome virus [J]. Gen Virol, 1998, 79:989-999
Plagemann P.G... GP5 ectodomain epitope of porcine reproductive and respiratory syndrome virus strain Lelystad virus [J]. Virus Res, 2004, 102(2):225-230
Plana D. J., Climent I. S. J., Alicla U., et al. Baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain Olot/91 .Involvement of ORF3 and ORFS proteins in protection. Virus Genes, 1997, 14(1): 19-29
Prechl J., Baiu D. C., Horvath A., et al. Modeling the presentation of C3d-coated antigen by Blymphocytes: enhancement by CR1/2-BCR co-ligation is selective for the coligating antigen. Int Immunol, 2002, 14(3): 241-247
Prota A. E., Sage D. R., Stehle T., et al. The crystal structure of human CD21: implications for Epstein- Barr virus and C3d binding. Pro Nat Aead Sei, 2002, 99: 10641-10646
Qazi K. R., Wikmna M., Vasconcelos N. M., et al. Enhancement of DNA Vaccine Potency by linkage of Plasmodium falciparum malarial antigen gene fused with fragment of HSP70 gene. Vaccine, 2005, 23(9): 1114-1125
Qiu H. J., Tian Z. J., Tong G.. Z., et al. Protective immunity induced by a recombinant pseudorabies virus expressing the GP5 of porcine reproductive and respiratory syndrome virus in piglets[J].Vet Immunol Immunopathol, 2005, 106:309-319
Rabinovich N.R., McInnes, Klein. Vaccine technologies: view to the future. Science, 1994, 256: 1401-1404
Reed S.G., Pihid L., et al. IL-1 as adjuvant: role of T cell in the augmentation of specific antibody production by recombinant human IL-1[J]. Immunol, 1989,142:3129-3133
Roberts C. W., Fergoson D. J. P., Jebbari H., et al. Different roles for IL-4 during the course of toxoplasma gondii infection. Infect Immunol, 1996, 64: 897-906
Robinson H. L., Webster R. G.. Protection against a lethal influenza challenge by immuniz- ation hem-agglutinin expressing Plasmid DNA. Vaccine, 1993, 11:957-960
Rodriguez E. R., Skojec D. V., Tan C. D., et al. Antibody-mediated rejection in human cardiac allografts: evaluation of immunoglobulins and complement activation productsC4d and C3d as markers. Am J Transplant, 2005, 5(11): 2778-2785
Rodriguez F., Harkins S., Redwine J. M., et al. CD4 (+) T cells induced by a DNA vaccine: immunological consequences of epitope-specific lysesmal targeting. J Virol, 2001, 75(21): 10421-10430
Rodriguez M. J., Sarraseca J., Fominaya J., et al. Identification of an immunodom- inant epitope in the C terminus of glycoprotein 5 of porcine reproductive and respiratory syndrome virus [J].J Gen Virol, 2001, 82(5): 995-999
Rolland A.P., Mumper R.J.. Plasmid delivery to muscle: Recent advances in polymer delivery systems. Adv. Drug.Deliv. Rev., 1998, 30:151-172
Rollier C., charollois C., Jamard C., et al. Maternally transferred antibodies form DNAimmunized avian Protect offspring against hepadnavirus infection. J Virol, 2000, 74(10):4908-4911
Ross T. M., Xu Y., Bright R. A., et al. C3d enhancement of antibodies to hemagglutinin accelerates protection against influenza virus challenge. Nat Immunol, 2000, 1(2): 127-131
Russell P. H., Mackie A.. Eye-drop DNA can induce IgA in the tears and bile of chickens. Vet Immune Immunopathol, 2001, 80(3):327~332
Sakaguchi M., Nakamura H., Soroda K., et al. Protection of chickens from Newcastle disease by vaccination with a liner Plasmid DNA expressing the F Protein of Newcastle disease virus. Vaccine, 1996, 14(8):747-752
Sanchez A. E., Aquino G., Ostoa Saloma P., et al. Cholera toxin B-subunit gene enhances mucosal immunoglobulin A, Thl-type, and CD8+ cytotoxic responses when co administered intradermally with a DNA Vaccine. Clin Diagn Lab Immunol, 2004, 11(4): 711-719
Sasaki S., Takeshita F.. Improvement of DNA vaccine immunogenicity by a dual antigen expression system. Biochem Biophys Res Commun, 2004, 315(1):38-43
Saxrias M. R., Franchini S., Canziani G.., et al. Kinetic analysis of the interactions of complement receptor 2(CR2, CD21) with its ligands C3d, iC3b, and the EBV glycoprotein gp350/220. J Immunol, 2001, 167(3): 1490-1499
Schultz J., Dollenmaier G., Moiling K.. Update on antiviral DNA vaccine research (1998-2000). Intervirology, 2000, 43(4-6):197-217
Seo S. H., Wang L., Smith R., et al. The carbosyl terminal 120-residue Polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and Protects chickens from acute infection. J. Virol, 1997, 71 (10): 7889-7894
Serge Sagodira, et al. Protection of kids cryptosporidium parvum infection after im- munization of dams with cp15-DNA [J].J vaccine, 1997, 17: 2346-2355
Shen S., Kwang J., Liu W., et al. Determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the Nsp2 gene with a unique insertion[J].Arch Virol, 2000, 145 (5):871-883
Suarez P., Diaz G.. M., Prieto C., et al. Open reading frame 5 of porcine reproductive andrespiratory syndrome virus as a cause of virus-induced apoptosis [J].Virol, 1996, 70:2876-2882
Suradhat S., Braun R. P., Lewis P. J., et al. Fusion of C3d molecule with bovine rotavirus VP7 or bovine herpesvirus type 1 glycoprotein D inhibits immune responses following DNA immunization. Vet Immunol Immunopathol, 2001, 83(1-2): 79-92
Szakony G., Guthridge J. M., Li D., et al.Structure of complement receptor 2 in complex with its C3d ligand. Science, 2001, 292: 1725-1728
Tang D. C., Johnson S. A.. Genetic immunization is a simple method for eliciting an immuneresponse. Nature, 1992, 356:152-154
Temperton N. J., Quenelle D. C., Lawson K. M., et al. Enhancement of humoral immune responses to a human cytomegalovirus DNA vaccine: adjuvant effects of aluminum Phosphate and CpG oligodeoxynucleotides. J Med Virol, 2003, 70(l): 86-90
Test S. T., Mitsuyoshi J., Commolly C. C., et al. Increased immunogenicity and induction of class switching by conjugation of complement C3d to pneumococcal serotype14 capsular polysaccharide.Infect Immun, 2001, 69(5): 3031-3040
Thornton B. P., Vetvicka V., Ross G. D.. Function of C3 in a humoral response: iC3b/C3dg bound to an immune complex generated with natural antibody and a primary antigen promotes antigen uptake and the expression of co-stimulatory molecules by all B cells, but only stimulates immunoglobulin synthesis by antigen-specific B cells. Clin Exp Immunol, 1996, 104(3): 531-537
Toapanta F. R., Ross T. M.. Mouse strain-dependent differences in enhancement of immune responses by C3d. Vaccine, 2004, 22(13-14): 1773-1781
Tong T., Fan H., Tan Y., et al. C3d enhanced DNA vaccination induced humoral immune response to glycoprotein C of pseudorabies virus. Biochem Biophys Res Commun, 2006, 347(4):845-851
Torres C. A., Iwasaki A., Barber B. H., et al. Differential dependence on target site tissue for gene gun and intramuscular DNA immunizations [J]. Immunol, 1997, 158(10): 4529- 4532
Ulmer J. B., et al. Science, 1993, 259: 1745-1749
Ulmer J. B., Donnelly J J, Liu M A. Toward the development of DNA vaccines. Curr Opin Bio techno, 1996, 7(6): 653-658
Vahlsing H. L., Yancauckas M. A., Sawdey M., et al. Immunization with plasmid DNA using a pneumatic gun. J Immunol Methods, 1994, 175: 11-22
Vanrompay D., Vanloovk M., Cox E., et al. Genetic immunization for Chlamydia Psitlaci. Verh K Acad Geneeskd Belg, 2001, 63(2):177-188
Veronique F. B., Kolb J. P., Sabine R., et al. Functional properties of soluble CD21. Immuno Pharrnaeology, 1999, 42: 31-37
Villiers C. L., Lefebvre N., Villiers M. B., et al .Role of C3 fragments in the control of the introcellular antigen processing [J]. Mo. Immunol, 1996, 33: 1
Vitadello M.. Gene transfer in regenerating muscle. Hum Gene Ther, 1994, 5:11-18 Wang B., Boyer J., Srikantar V., et al. DNA inoculation induces neutralizing immune responses against human immuno deficiency virus type in mice and nonprimates. Virol, 1995, 208: 827-831
Wang B., Dang K., Agadjanyan M.G.., et al. Mucosal immunization with a DNA vaccine induces immune responses against HIV-1 at a mucosal site. Vaccine, 1997, 15:821-825
Wang B.,Ugen K. E., Srikantan V.,et al. Gene inoculation generates immune responses against human immunodeficiency virus type 1 [J].Proc Natl Acid Sci: USA, 1993,90 (9): 4156-4160
Wang L., Sunyer J. O., Bello L. J.. Fusion to C3d enhances the immunogenicity of the E2 lycoprotein of type 2 bovine viral diarrhea virus. Virology, 2004, 78(4): 1616-1622
Wang X. L., Li D. J., Yuan M. M., et al. Enhancement of humoral immunity to the hCG beta Protein antigen by fusing a molecular adjuvant C3d3. J Re Prod Immunol, 2004, 63: 97-110
Wang Y. B., Li Y. J., Zhang F. C.. Enhancing the induced immune responses of lagurus zona pellucida 3 DNA vaccines by molecular adjuvant C3d [J]. Fen Zi Sheng Wu Xue Bao, 2006, 39(4):293-303
Weiland E., Wieczorek K. M., Kohl D., et al. Monoclonal antibodies to the GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the GP4[J]. Vet Microbiol. 1999, 66(3):171-186
Wensvoort G.. Lelystad virus and the porcine epidemic abortion and respiratory syndrome [J].Vet Res. 1993, 24:117-124
Whalen R. G...DNA sequences necessary for effective intradermal gene immunization. Clinical Immunol and Immunopath, 1995, 75:1-12
Whitton J. L., Rodriguez F., Zhang J., et al. DNA immunization: mechanistic studies. Vaccine, 1999, 17(1314):1612-1619
Wissink E. H., Kroese M. V., Van Wijk H. A., et al. Envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus [J]. J Virol, 2005, 79(19): 12495-12506
Witte S.B., Chard B. C., Loughin T. A., et al. Development of a recombinant nucleoprotein- based enzyme-linked innunosorbentassay for quantification of antibodies against porcine reproductive and respiratory syndrome virus[J].Clin Diagn Lab Immunol. 2000, 7(4):199-203
Wolff J A., Malone R. W., Williams P., et al. Direct gene transfer into mouse muscle in vivo. Science, 1990, 247:1465-1468
Wolff J. A., Malone R. W., Williams P., et al. Intruductions of foreign genes into tissues of living mice by DNA-coated microprojectiles[J].Proc Natl Acid Sci,1991,88(7): 2726-2730
Wootton S., Koljesar G.., Yang L., et al. Antigenic importance of the carboxy-terminal beta-strand of the porcine reproductive and respiratory syndrome virus nucleocapsid protein[J].Clin Diagn Lab Immunol, 2001, 8(3):598-603
Xiang Z. Q., Soitalink S. M., Tran., et al. Vaccination with a plasmid vector carrying the rabiesvirus goycoprotein gene induces protective immunity against rabiesvirus. Virology, 1994, 199:132-140
Xue Q., Zhao Y. G.., Zhou Y. J., et al. Immunization with plasmids encoding porcine reproductive and respiratory syndrome virus ORFs5 and 7,and porcine IL-2 and IFN-gamma[J]. Vet Immunol Immunopathol, 2004, 102:291-298
Yang L., Frey M. L., Yoon K. J., et al. Categorization of North American porcine reproductive and respiratory syndrome virus: epitopic profiles of the N, M, GPS and GP3 proteins and susceptibility to neutrolization. Arch Virol, 2000, 145(8): 1599-1619
Yao X. Y., Wang H. M., Li D. J., et al. Inoculation of Lactobacillus expressing hCGβin vagina induces anti-hCGβantibody response at murine vaginal mucosal. J Re Prod Immunol, 2004, 63: 111-122
Yokoyama M., Hassett D. E., Zhang J., et al. DNA immunization can stimulate florid local inflammation, and the antiviral immunity induced varies depending on injection site. Vaccine, 1997, 15(5):553-560
Yoon K. J., Wu L. L., Zimmeran J. J., et al. Field isolates of porcine reproductive and respiratory syndrome virus vary in their susceptibility to antibody dependent enhancement (ADE) of infection [J].Vet Microbiol, 1997, 55:227-287
Young K. R., Teal B. E., Brooks Y., et al. Unique v3 loop sequence derived from the R2 strain of HIV type 1 elicits broad neutralizing antibodies. AIDS Res Hum Retroviruses, 2004, 20(11):1259-1268
Zhu Y., Ren J., Da’dara A., et al. The Protective effect of a schistosoma japonicum Chinese strain 23Dka plasmid DNA Vaccine in pigs is enhanced with IL-12. Vaccine, 2004, 15, 23(1): 78-83