单纯疱疹病毒Ⅱ型糖蛋白D胞外区片段在哺乳动物细胞中的表达及其免疫活性  

Expression of Excellular Domain of Glycoprotein D of Herpes Simplex Virus Type 2 in Mammalian Cells and Immunologic Activity of Expressed Product

作　　者：管文燕[1] 王正茂[1] 李琳[1] 李越希[1,2] 

机构地区：[1]南京医科大学基础医学院生物化学与分子生物学系,南京210029 [2]南京军区军事医学研究所,南京210002

出　　处：《中国生物制品学杂志》2011年第4期384-388,共5页Chinese Journal of Biologicals

基　　金：国家"863"计划项目(2006AA02A226)

摘　　要：目的在哺乳动物细胞中表达单纯疱疹病毒Ⅱ型(Herpes simplex virus type-2,HSV-2)糖蛋白D(GlycoproteinD,gD)胞外区片段,并分析其免疫活性。方法化学合成HSV-2 G株gD蛋白胞外区片断的基因序列gDt,以pCEP4作为表达载体,构建N-末端带His标签的重组表达质粒pCEP4-gDt,转染至HEK293细胞进行表达。表达的蛋白采用镍离子柱亲和层析纯化,ELISA法检测目的蛋白的抗原性。以纯化的重组蛋白免疫小鼠制备多抗血清,间接ELISA法检测效价。结果重组表达质粒经PCR、双酶切和DNA测序证实构建正确;表达产物经Western blot分析,在相对分子质量约46 000处可见目的蛋白条带;纯化的重组蛋白浓度约为45μg/ml,经ELISA检测证实具有良好的抗原性,免疫小鼠5周后,血清中特异性抗体效价达5×103。结论已在哺乳动物细胞中表达了HSV-2 gD胞外区片段,其具有良好的抗原性和免疫原性,为HSV基因重组亚单位疫苗的研制奠定了基础。Objective To express the excellular domain of glycoprotein D（gD） of herpes simplex virus type 2（HSV-2） in mammalian cells and analyze the immunologic activity of expressed product.Methods The gDt gene sequence encoding excellular domain of gD of HSV-2 G strain was chemically synthesized and inserted into vector pCEP4.The constructed recombinant plasmid pCEP4-gDt,with a His label at N-terminus,was transfected to HEK293 cells for expression.The expressed protein was purified by nickel ion column affinity chromatography and tested for antigenicity by ELISA.Polyclonal antisera were prepared by immunizing mice with the purified recombinant protein and determined for titer by ELISA.Results PCR,restriction analysis and DNA sequencing proved that recombinant plasmid pCEP4-gDt was constructed correctly.Western blot showed a target protein band with relative molecular mass of about 46 000.The purified recombinant protein,at a concentration of about 45 μg/ml,showed good antigenicity as proved by ELISA,which induced specific antibody titer of 5 × 103 in the sera of mice 5 weeks after immunization.Conclusion The excellular domain of gD of HSV-2 was expressed in mammalian cells,and showed good antigenicity and immunogenicity,which laid a foundation of preparation of recombinant subunit vaccine against HSV.

关 键 词：单纯疱疹病毒 包膜糖蛋白D 哺乳动物细胞 免疫活性 

分 类 号：R373.11[医药卫生—病原生物学]