摘要
筛选了3个位于流行性乙型脑炎病毒(JEV)E蛋白上不同位置的抗原表位区基因,利用重叠PCR方法分别与猪细小病毒(PPV)VP2基因相连。将目的基因定向插入真核表达载体pcDNA3.1(+),构建了重组质粒pcDNA3.1-EA-VP2、pcDNA3.1-EB-VP2、pcDNA3.1-EC-VP2。将重组质粒在无免疫佐剂参与的情况下免疫BALB/c小鼠,同时设空质粒接种组及PBS缓冲液接种组作为空白对照,采用ELISA法、淋巴细胞增殖试验和流式细胞仪技术分别对抗体水平、脾淋巴细胞转化功能和小鼠外周血中CD3+、CD4+和CD8+T淋巴细胞数进行检测。结果显示,重组质粒pcDNA3.1-EA-VP2、pcDNA3.1-EB-VP2、pcDNA3.1-EC-VP2接种小鼠后,都能诱导小鼠产生JEV、PPV特异性抗体,并且促进脾淋巴细胞增殖。试验组与对照组比较差异极显著(P<0.01);试验组小鼠CD3+、CD4+T淋巴细胞数在首疫后第7天高于对照组(P<0.01),CD8+T淋巴细胞数在首免后第14天高于对照组(P<0.01)。pcDNA3.1-EB-VP2免疫组小鼠的体液免疫和细胞免疫应答水平均高于pcDNA3.1-EA-VP2和pcDNA3.1-EC-VP2免疫组。表明,用JEV E蛋白抗原表位区基因与PPV VP2基因构建的真核表达质粒能够诱导小鼠产生良好的细胞免疫和体液免疫应答。
Three different epitopes of Japanese encephalitis virus（JEV）E protein were chos en to be combined to porcine!parvovirus（PPV）VP2 gene by overlapping extension P CR methods,respectively.The three fused genes were cloned into pcDNA3.1（＋）the recombinant eukaryotic ex pression plasmids were named pcDNA3.1-EA-VP2,pcDNA3.1-EB-VP2 and pcDNA3.1-EC-VP2,respectively. Mice were injected intramuscularly with recombinant plasmids without any adjuvan t,and two control groups were injected with pCDNA3.1（＋）and PBS,respectively.To evaluate the immune efficacy,IgG was determined by ELISA,MTT assay and flow cytometry sorting were used to determine the subsets of T lym phocyte.The results indicated that the antibody levels resulted from the recombinant pla smid and the reaction of the splenic T lymphocytes to the ConA stimulation were significantly different between all experiment groups and the controls（P〈0.01）.The numbers of CD3＋ and CD4＋ T-lymphocytes of the e xperiment groups were significantly higher than that of the controls on day 7 af ter 1st-vaccination.The numbers of CD8＋ T-lymphocytes of experiment groups were higher than that of the control groups on day 14 after 1st-vaccination.The mice immunized with pcDNA3.1-EB-VP2 induced higher levels of cellula r and humoral immune responses than the other two experiment groups.These results indicated that eukaryotic expression plasmids with antigenic deter minant of JEV E gene and PPV VP2 gene were able to induce satisfactory humoral a nd cellular immune responses in mice.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2010年第9期900-906,共7页
Chinese Veterinary Science
基金
国家自然科学基金项目(30500019)
四川省教育厅重点实验室项目(07ZZ027)
教育部"长江学者和创新团队发展计划"创新团队项目(IRT0848)
