【作者】 张德庆；

【导师】 牛钟相；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2011， 博士

【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。更多还原

【Abstract】 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.更多还原