基因Ⅰ型乙型脑炎病毒病毒样颗粒的制备及其对小鼠的免疫原性分析

Generation of GenotypeⅠJapanese Encephalitis Virus Virus-like Particles and Evaluation of Its Immunogenicity in Mice

降低猪场的流行性乙型脑炎(Japanese encephalitis,JE)感染率对于阻断人的JE暴发至关重要,鉴于我国JE防控形势的紧迫性和现有基因Ⅲ型商品化乙型脑炎病毒疫苗对部分基因Ⅰ型乙型脑炎病毒(genotypeⅠJapanese encephalitis virus,GⅠJEV)流行毒株免疫保护的不确定性,急需研制安全高效、成本低廉的新型猪用GI JEV疫苗。本研究在杆状病毒载体pFastBacTM多克隆酶切位点插入GⅠJEV的prME基因表达盒,测序验证后的阳性重组质粒转染DH10BacTM感受态细胞后获得Bacmid-prME重组杆粒,进一步将其转染至Sf9昆虫细胞后,获得Bac-prME重组杆状病毒。Western blot、间接免疫荧光及电镜观察GⅠJEV病毒样颗粒组成蛋白表达及病毒样颗粒形成情况,通过小鼠免疫试验初步评价GⅠJEV病毒样颗粒的免疫原性。Sf9昆虫细胞中prME基因表达盒编码的蛋白得到高效表达,且组装形成病毒样颗粒。病毒样颗粒免疫原性评估结果表明:GⅠJEV病毒样颗粒可诱导免疫小鼠产生高滴度的乙型脑炎病毒中和抗体。本研究利用杆状病毒昆虫细胞表达系统获得了GⅠJEV病毒样颗粒,为研制安全高效的乙型脑炎病毒病毒样颗粒亚单位疫苗提供物质基础。

Decreasing the circulation of the Japanese encephalitis virus(JEV)in pigs is critical for blocking JEV infection in human.In view of the urgency of JE prevention and control in China and the uncertainty of the commercial GenotypeⅢJEV attenuated vaccine to the immuno-protection of partial GenotypeⅠJEV(GⅠJEV)endemic strains,it is urgent to develop a safe,effective and low-cost GⅠJEV vaccine for pigs.The expression cassette of JEV prME gene was cloned into the baculovirus shuttle vector to generate a recombinant bacmid(Bacmid-prME),the recombinant baculovirus was produced by transfecting Sf9 cells with Bacmid-prME.The accurate expression of the recombinant prME protein was confirmed by Western blot and immunofluorescence analysis,and the formation of Virus-Like Particles(VLPs)was verified through observing under Electronic microscopy.The immunogenicity of GⅠJEV VLPs was further evaluated in mice.The recombinant prME protein was efficiently expressed in Sf9 cells and assembled into VLPs.GⅠJEV VLPs induced the higher neutralizing antibody titer in mice.In our study,GⅠJEV VLPs obtained in the baculovirus expression system provides a material basis for the development of a safe and effective JEV subunit vaccine.