热休克蛋白70-Ⅱ型单纯疱疹病毒糖蛋白DDNA疫苗的构建及表达  

Construction and expression of Hsp70-HSV2gD DNA vaccine

作　　者：樊建勇[1] 杨慧兰[1] 关蕾[1] 王颖[1] 仕瑶慧[1] 

机构地区：[1]广州军区广州总医院皮肤科,广东广州510010

出　　处：《医学研究生学报》2007年第4期349-352,I0001,共5页Journal of Medical Postgraduates

基　　金：国家自然科学基金资助项目(批准号:30371290);广东省医学科研基金资助项目(批准号:A2006492)

摘　　要：目的:针对Ⅱ型单纯疱疹病毒(HSV-2)包膜糖蛋白D(gD),以热休克蛋白70(Hsp70)为载体,构建pVAX-Hsp70-HSV2gD DNA疫苗。方法:将Hsp70和HSV-2gD蛋白的基因分别克隆至真核表达载体pVAX,构建成重组质粒pVAX-Hsp70-gD并测序鉴定。重组质粒pVAX-Hsp70-gD转染COS-7细胞,用免疫组化、SDS-PAGE和W est-ern b lotting方法鉴定重组质粒的表达情况。结果:测序证实重组质粒序列正确,表达产物的SDS-PAGE分析发现,在相对分子量为92 000处有外源蛋白表达,与预期蛋白带一致。免疫组化方法和W estern b lotting也证明,构建的重组质粒能在COS-7细胞内表达。pVAX-Hsp70-HSV2gD组核酸疫苗免疫的小鼠,其脾淋巴细胞培养上清中γ-干扰素的水平高于其他组(P<0.05)。结论:成功构建了pVAX-Hsp70-HSV2gD DNA疫苗,为其进一步的研究打下了基础。Objective： To Construct and express pVAX-Hsp70-HSV2gD DNA Vaccine. Methods： Genes of Hsp70 and HSV-2gD were subcloned into vectors pVAX respectively. After confirmed by DNA sequence analysis, the recombinant plasmid pVAX-Hsp70-HSV2gD was transformed into COS-7 cells. The expressed protein was characterized by immunohistochemistry, SDS-PAGE and Western blot. Re- sults：Recombinant plasmids were confirmed by DNA sequencing. The expressed protein analyzed by SDS-PAGE showed a new band with an apparent molecular mass corresponding to the predicted size （92 000）. Immunohistochemistry and Western Blotting analysis demonstrated that gD protein was expressed in COS-7 cells. The level of γ-IFN in culture of pVAX-HspTO-HSV2gD group was obviously higher than those of other groups （ P 〈 0.05, respectively ）. Conclusion： The expression of the pVAX- HspTO-HSV2gD DNA Vaccine was successful and laid a solid foundation for its further research.

关 键 词：热休克蛋白70 糖蛋白D 单纯疱疹病毒 DNA疫苗 

分 类 号：R511[医药卫生—内科学]