日本乙型脑炎病毒PrMΔE与tPA信号肽融合基因DNA疫苗的研究  

Study on DNA vaccine based on fused gene containing PrM ΔE gene of Japanese encephalitis virus and signal peptide sequence of tPA

作　　者：刘凯[1] 孟丽军[1] 张亮[1] 黄小波[1,2] 文心田[1,2,3] 曹三杰[1,2] 

机构地区：[1]四川农业大学预防兽医研究所猪病研究中心,四川雅安625014 [2]四川农业大学动物医学院人兽共患病研究室,四川成都611130 [3]农业部兽用药物与兽医生物技术四川科学观测试验站,四川雅安625014

出　　处：《中国兽医科学》2013年第4期351-357,共7页Chinese Veterinary Science

基　　金：公益性行业(农业)科研专项经费项目(201203082)

摘　　要：采用RT-PCR和重叠PCR方法分别扩增出日本乙型脑炎病毒PrMΔE基因和tPA信号肽序列,并将其插入载体pVAX1,将纯化后的重组质粒转染COS-7细胞,用间接免疫荧光法检测质粒在细胞中的瞬时表达情况,并用制备的质粒免疫昆明小鼠,检测其特异性血清IgG抗体水平、促脾淋巴细胞增长率及攻毒保护率。结果显示,真核表达质粒pVAX-tPA-PrMΔE构建成功;间接免疫荧光试验显示,该质粒在COS-7细胞中的表达产物可与鼠抗日本乙型脑炎病毒单克隆抗体发生特异性反应,tPA信号肽序列能明显增强PrMΔE基因DNA疫苗的免疫原性,显著提高小鼠的体液免疫和细胞免疫水平,能强有力地抵抗强毒株CZ-1的攻击。结果表明,成功构建了日本乙型脑炎病毒PrMΔE基因与tPA信号肽序列融合基因DNA疫苗(pVAX-tPA-PrMΔE),为日本乙型脑炎病毒基因工程疫苗的进一步研究及应用奠定了基础。To study the immune efficacy of a DNA vaccine with PrMAE gene of Japanese encephalitis virus（JEV） and signal peptide sequence of tissue plasminogen activator（tPA）,the PrMAE gene and signal peptide sequence of tPA were amplified by RT-PCR and overlap PCR, respectively. The two gene fragments were inserted into the expression vector pVAX1. The expression of the plasmid pVAX-tPA-PrMAE in COS-7 cells was identified. Kunming mice were inoculated with plasmid pVAX-tPA-PrMAE,after first im- munization,the sera were collected to examine anti-JEV IgG antibody level by ELISA and splenic lympho- cytes were detected by MTT. Vaccinated mice were challenged with JEV CZ-I（100 TCIDs0/0.1 mL ） in intracerebral inoculations. In result, the signal peptide sequence of tPA was able to improve the immunogenieity of the DNA vaccine, and to enhance the humoral immunity and cellular immunity of the vaccinated mice. In conclusion,this study constructed a new DNA vaccine of JEV,which paves the way for the further study and application of JEV genetic engineering vaccine.

关 键 词：日本乙型脑炎病毒 PrMΔE基因 tPA信号肽序列 DNA疫苗 免疫原性 

分 类 号：S852.659.6[农业科学—基础兽医学]