日本乙型脑炎病毒强、弱毒株非结构蛋白NS1、NS2A和NS1-NS2A诱导小鼠CD4^+T细胞应答的研究

Japanese Encephalitis Virus Virulent Strains and Attenuated Strains of Non-Structural Proteins NS1,NS2A and NS1-NS2A Induce Mouse CD4^+ T Cell Responses

为了解日本乙型脑炎病毒(JEV)强、弱毒株非结构蛋白的免疫效果,笔者分析了JEV强、弱毒株非结构蛋白NS1、NS2A和NS1-2A真核表达的差异。通过观察JE减毒活疫苗(SA14-14-2株)和JEV贵州分离株(GZ株)分别在BHK-21细胞上出现的CPE,并收集强、弱毒株的细胞悬液,提取总RNA,分别设计6对特异性引物,应用RT-PCR技术扩增非结构蛋白NS1、NS2A和NS1-2A的编码基因,构建真核表达质粒pcDNA3.1(+)-NS1r、pcDNA3.1(+)-NS2Ar、pcDNA3.1(+)-NS1-2Ar、pcDNA3.1(+)-NS1q、pcDNA3.1(+)-NS2Aq和pcDNA3.1(+)-NS1-2Aq,将构建成功的6种真核表达质粒免疫健康小鼠,最后采集小鼠的血液和组织分别进行JEV抗体检测和细胞免疫水平检测。研究结果显示:JEV GZ强毒株与JEV SA14-14-2弱毒株能在BHK21细胞上增殖并产生CPE现象;构建的6种真核表达质粒免疫小鼠后经血常规和T淋巴亚群检测可以极显著或显著增加CD4+和CD8+细胞的含量。表明JEV强毒株非结构蛋白所诱导的细胞免疫略优于弱毒株非结构蛋白诱导的细胞免疫,但差异不明显。本研究结果可为日本乙型脑炎病毒的病原基础研究提供参考。

In order to understand the immunological effect of nonstructural protein of Japanese encephalitis virus（JEV）,we analyzed the eukaryotic expression differences of NS1,NS2 A and NS1-2 A,nonstructural proteins of JEV.CPE was observed on BHK-21 cells by observing JE live attenuated vaccine（SA14-14-2 strain）and JEV Guizhou isolate（GZ strain）respectively.Cell suspensions of virulent,attenuated strains were collected and their total RNA were extracted.The NS1,NS2 Aand NS1-2 Agenes were amplified by RT-PCR,and six eukaryotic expressionplasmids,pcDNA3.1（＋）-NS1 r,pcDNA3.1（＋）-NS2 Ar,pcDNA3.1（＋）-NS1-2 Ar,pcDNA3.1（＋）-NS1 q,pcDNA3.1（＋）-NS2 Aq and pcDNA3.1（＋）-NS1-2 Aq,were constructed successfully.The healthy mice were immunized with the above plasmids,and the blood and tissues of those mice were collected for JEV antibody detection and cell immunoassay respectively.The results showed that JEV GZ virulent strain and JEV SA14-14-2 virulent strain could proliferate and produce CPE on BHK21 cells;The results of routine blood tests and T lymphocyte subsets detection showed that the 6 eukaryotic expression plasmids immunization in mice increased the levels of CD4＋and CD8＋cells significantly or extremely significantly.The results indicated that the cellular immunity produced by the non-structural proteins of the attenuated strain JEV was slightly better than that produced by the non-structural proteins of the attenuated strains,but the difference was not significant.These results provide reference for the basic research of encephalitis virus pathogens.