细粒棘球蚴95抗原在真核昆虫表达系统中表达、纯化及免疫原性初步分析

Expression and Purification of Recombinant Protein Echinococcus Granulosus 95 in Eukaryotic Insect Expression System and the Preliminary Analysis on its Immunogenicity

目的采用真核昆虫表达系统表达细粒棘球蚴95(Eg95)抗原,探讨其免疫原性。方法从GeneBank获取Eg95基因序列(序列编号:EU595937.1),合成开放读码框(CDS)全长基因;利用杆状病毒表达系统Bac-to-Bac symtem在昆虫细胞中表达目的基因Eg95,亲和层析纯化,Western blot鉴定;对Balb/c小鼠分别免疫接种重组昆虫表达目的蛋白Eg95(简称eu rEg95)、原核重组表达Eg95(简称pro rEg95)、阴性对照PBS,制备免疫血清,间接法ELISA对各组特异性IgG抗体进行分析。结果合成Eg95基因CDS全长471 bp,经测序与理论完全一致;Western blot证实目的蛋白通过重组杆状病毒在昆虫细胞内实现可溶性表达;ELISA结果表明:eu rEg95能够诱导产生原头蚴特异性IgG抗体,抗体水平高于pro rEg95组(P<0.05)。结论利用昆虫杆状病毒表达系统成功表达出具有良好免疫原性的Eg95抗原,为优化细粒棘球蚴疫苗奠定基础。

Objective To generate soluble recombinant Eg95 antigen using eukaryotic insect expression system and analyze its immunogenicity. Methods The whole open reading frame of Eg 95 gene was obtained from Genebank( sequence number: EU595937. 1) and was expressed in insect cells by Bac-to-Bac system. Using affinity chromatography,purified protein was obtained,which was identified by Western blot.Then Balb /c mice were vaccinated by eukaryotic recombinant Eg95( eu rEg95),prokaryotic recombinant Eg95( pro rEg95) or PBS. Mice sera were detected for IgG value( OD450) by indirect ELISA. Results Eg95 gene sequence was synthesized correctly. Western blot identified that the target gene expressed successfully in insect expression system as soluble protein. In ELISA assay,purified eu rEg95 not only had capacity to induce effective specific IgG antibody response,but also induced more value of specific IgG than pro rEg95. Conclusion eu rEg95 was successfully expressed in insect expression system with efficient immunogenicity. This study is further benefit for the optimization of E. granulosis vaccine.