【作者】 石娜；

【导师】 任晓峰；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2010， 硕士

【摘要】 本实验分别将(猪繁殖与呼吸综合征病毒)PRRSV ORF5基因与猪IL-15基因亚克隆至pVAX1真核表达载体中,构建了两个重组真核表达质粒pVAX1-PRRSV ORF5和pVAX1-pIL-15。通过脂质体转染法将pVAX1-PRRSV ORF5和pVAX1-pIL-15转入Vero细胞中进行了表达,试验结果表明,两个基因都可以在体外进行表达,且表达的蛋白可以被PRRSV ORF5和pIL-15多克隆抗体所检测。以pVAX1-PRRSV ORF5和pVAX1-pIL-15重组真核表达质粒免疫小鼠,分为7个组,分别是pVAX1-PRRSV ORF5单独免疫组、pVAX1-pIL-15单独免疫组、pVAX1-PRRSV ORF5(100μg)与pVAX1-pIL-15(100μg)联合免疫组、pVAX1-PRRSV ORF5(100μg)与pVAX1-pIL-15(200μg)联合免疫组、pVAX1-PRRSV ORF5(100μg)与pVAX1-pIL-15(50μg)联合免疫组、pVAX1空载体对照组和PBS对照组。利用淋巴细胞转化试验、酶联免疫吸附试验、流式细胞仪技术对不同处理组小鼠的各项免疫指标进行了检测。试验结果表明,各免疫组小鼠的各项免疫指标均比pVAX1空载体对照组和PBS对照组高,加入pIL-15的结果尤为显著。在淋巴细胞转化试验和CD4+、CD8+细胞数量的变化及小鼠血液中IFN-γ的检测试验中,加pIL-15中剂量(100μg)组的效果最为明显,其次是加pIL-15小剂量(50μg )组,加pIL-15大剂量(200μg)组的效果差于以上两组,但也比pVAX1-PRRSV ORF5单独免疫组效果明显。而酶联免疫吸附试验检测抗PRRSV ORF5 IgG的结果显示,加pIL-15大剂量(200μg)组的免疫效果优于其他各组。同时在不同时间点收集组织样品,通过PCR检测pVAX1-PRRSV ORF5质粒在小鼠体内的动态分布情况。结果表明,肌肉注射核酸疫苗后,pVAX1-PRRSV ORF5迅速分布到小鼠其他组织器官中,在免疫后3 h即可在脑组织、心脏、肝脏、肺脏、肾脏中检测到,一直持续到免疫后42天。本实验还构建了二个pGEX-6p-pIL-15原核表达质粒,二个质粒分别缺失了pIL-15 N端29 aa和48 aa信号肽序列。在大肠杆菌中获得了高效表达的pIL-15蛋白。纯化后蛋白免疫新西兰大白兔获得兔源多克隆抗体。酶联免疫吸附试验和免疫荧光结果表明pIL-15蛋白与多克隆抗体均具有生物学活性。综上,本研究首次将pIL-15基因与PRRSV ORF5基因质粒联合免疫。实验结果表明pIL-15基因作为PRRSV ORF5抗原基因分子佐剂,不但能促进机体的细胞免疫应答,而且能够提高机体体液免疫应答,进而提高了机体的免疫反应。为进一步研究pIL-15生物活性提供了数据,同时为新型免疫增强型抗病毒疫苗的研究提供了理论基础。更多还原

【Abstract】 In this study, PRRSV ORF5 gene and porcine IL-15 gene were inserted into the eukaryotic expression vector, pVAX1, resulting in recombinant plasmids, pVAX1-PRRSV ORF5 and pVAX1-pIL-15. The plasmids were transfected into Vero cells and the results showed that the transient expression of both genes was confirmed by respective antibodies.Experimental mice were divided into 7 groups. They were pVAX1-PRRSV ORF5 group, pVAX1-pIL-15 group, pVAX1-PRRSV ORF5 (100μg) and pVAX1-pIL-15 (100μg) group, pVAX1-PRRSV ORF5 (100μg) and pVAX1-pIL-15 (200μg) group, pVAX1-PRRSV ORF5 (100μg) and pVAX1-pIL-15 (50μg) group, vector pVAX1 control group and PBS control group. Lymphocyte proliferative assay, enzyme linkered immunosorbent assay and flow cytometric technique were used to analyze the immune response of different groups.The results showed that the general immune responses of five immunized groups are higher than the empty pVAX1 vector and PBS control groups. Especially, the mice inoculated with the pVAX1-PRRSV ORF5 and pVAX1-pIL-15 had higher immune response than other groups in terms of the T lymphocyte proliferative function and the number of CD4+ and CD8+ in peripheral blood and spleen as well as the levels of IFN-γin peripheral blood. In addition, the pVAX1-pIL-15 (100μg) group had the highest cellular immune responses. The pVAX1-pIL-15 (50μg) group is better than the pVAX1-pIL-15 (200μg) group in terms of the stimulation of cellular immune responses. However, the highest level of specific anti-PRRSV ORF5 antibody in peripheral blood of immunized mice was stimulated by the combinaiton of the pVAX1-PRRSV ORF5 (100μg) and pVAX1-pIL-15 (200μg). These results are helpful for analyzing the adjuvant function of pIL-15.After inoculating different doses plasmids of pVAX1-PRRSV ORF5 and pVAX1-pIL-15 into the mice, tissue samples from different time points were collected and subjected to PCR amplifying PRRSV ORF5 gene. The results showed that the ORF5 gene is detectable shortly in mice after intramuscular injection, and it mainly can be detected in brain, heart, liver, lung and kidney from 3 h to 42 d post-immunization.At the same time, we designed two paris of primers to amplify two truncated pIL-15 genes with deletion of either N-terminal 29 aa signal peptide or N-terminal 48 aa signal peptide. The maximum expression of the pIL-15 was determined after cloning and expression of both genes in E.coli. The pIL-15 protein was used as an immunogen to inoculate a rabbit for generation of specific polyclonal antiserum. Our results showed that both the pIL-15 and its antiserum are biologically active.To sum up, our results indicate that pIL-15 can enhance the immune responses of DNA plasmid encoding the PRRSV ORF5 in mice. The optimal dose of the molecular adjuvant, pIL-15, is important for improvement of the DNA vaccination of PRRSV ORF5. The current study provide useful data for rational immunization of PRRSV ORF5 DNA.更多还原