真核细胞表达单纯疱疹病毒Ⅰ型包膜糖蛋白B及其抗原性与免疫原性分析

Expression of Herpes Simplex Virus Type Ⅰ Glycoprotein B in Eukaryotic Cells and Analysis of Its Antigenicity and Immunogenicity

为在真核细胞中表达并纯化I型单纯疱疹病毒(HSV I)包膜糖蛋白gB,并分析其抗原性和免疫原性,化学合成了包膜糖蛋白gB1胞外区基因片段,构建真核表达载体,并转染至HEK293细胞,表达的蛋白用羊抗HSV1+HSV2血清作为一抗,用ELISA检测其抗原性;用纯化的gB1蛋白免疫昆明小鼠,观察诱发抗体产生的时间及其效价,并用ELISA和Western blot检测小鼠抗gB1多克隆抗体特异性识别重组gB1抗原的能力,评价其免疫原性。结果显示在HEK293细胞中成功表达重组gB1蛋白,ELISA证实羊抗HSV1+HSV2多抗能够识别重组gB1蛋白;重组gB1蛋白免疫小鼠7周后,小鼠血清中多克隆抗体效价达到5×103,表明在真核细胞中高效表达并纯化的重组gB1蛋白具有良好的抗原性和免疫原性,为HSV检测试剂和疫苗研究提供了理论基础。

To express and purify herpes simplex virus type I（HSV I）glycoprotein B（gB）in eukaryote cells with the purpose of analyzing the antigenicity and immunogenicity.At the beginning,the extracellular domain fragment gene of gD1 was synthesized by chemical method and cloned into eukaryotic expression vector pCEP4 to construct the recombinant plasmid pCEP4-gD1.After transfection of HEK293 cells with the recombinant plasmid,the expressed protein was characterized by Western blot and purified through Ni affinity chromatography.Then antigenicity of the protein was detected by ELISA.Finally,the purified protein was used to immunize Kunming mice in 1,3,5 weeks respectively,and antiserum were collected in 3,5 and 7 weeks.The antibody titer was detected by indirect ELISA for immunogenicity analysis.Gene sequencing analysis demonstrates that the recombinant plasmid pCEP4-gB1 was constructed successfully.Western blot analysis indicated one major protein band,which molecular weight is approximate 85 kDa corresponding to the truncated forms of gB1 protein,was observed.In addition,ELISA detection showed that expressed gB1 has good antigenicity.After the third immunization,antibody titer of the mouse anti-gB1 was 5×103.The successful expression of the recombinant protein gD1,which can induce humoral immune response,lays a foundation for serological diagnosis and vaccine study of HSV.