单纯疱疹病毒2gD-Hsp70融合蛋白基因的构建及表达  

Construction and Expression of HSV-2gD-Hsp70 Fusion Protein Gene

作　　者：樊建勇[1] 杨慧兰[1] 王颖[1] 关蕾[1] 

机构地区：[1]广州军区广州总医院皮肤科,广州510010

出　　处：《生物工程学报》2006年第6期914-918,共5页Chinese Journal of Biotechnology

基　　金：国家自然科学基金资助项目(No.30371290);广东省医学科研基金资助课题(No.A2006492).~~

摘　　要：构建并原核表达Hsp70-HSV2gD融合蛋白。将Hsp70和HSV-2gD蛋白基因分别克隆到原核表达载体pGEX-4T-1,构建成重组质粒pGEX-4T-Hsp70-gD,并测序鉴定。重组质粒pGEX-4T-Hsp70-gD转化大肠杆菌DH5α后,IPTG诱导表达并进行SDS-PAGE分析。表达产物纯化后做Westernblot检测。将其肌注免疫BALB/c小鼠,检测融合蛋白对免疫小鼠脾淋巴细胞增殖、γ-干扰素产生以及血清中gDIgG水平的影响。表达产物的SDS-PAGE分析发现,在相对分子量为118kD处有外源蛋白表达,与预期蛋白带一致。用GST柱得到了纯化的Hsp70-HSV2gD融合蛋白。Westernblot证实,表达产物具有良好的活性。GST-Hsp70-gD组蛋白疫苗免疫的小鼠,其脾淋巴细胞刺激指数和脾淋巴细胞培养上清中γ-干扰素的水平高于其它组(P<0.05)。血清单纯疱疹病毒-2gD蛋白的抗体水平高于其它组(P<0.05)。To construct and express Hsp70-HSV2gD fusion protein. Genes of Hsp70 and HSV-2gD were subcloned into vectors pGEX-4T-1 respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pGEX-4T-HSP-gD was transformed into E. coli DH5α and induced to express with IPTG. The expressed protein was characterized by SDS-PAGE and Western blot after purified. BALB/c mice were immunized with fusion proteins respectively via intra-m uscular injection. The proliferation of spleen lymphocytes, the level of γ-IFN in culture and anti-HSV-2gD IgG antibody in serum was detected was detected. The expressed protein was analyzed by SDS-PAGE after induced with IPTG, which showed a new band with an apparent molecular mass corresponding to the predicted size（118kD）. Western Blotting analysis demonstrates that the purified Hsp70-HSV2gD fusion protein had specific binding activity. The stimulation indexes of spleen lymphocytes, the level of γ-IFN in culture and anti-HSV-2gD IgG antibody in serum of GST-Hsp70-gD group was obviously higher than that of other groups （ P 〈 0.05 respectively） . The successful expression of the Hsp70-HSV2gD fusion protein, which can induce immune responses, laid a solid foundation for its further research.

关 键 词：热休克蛋白70 gD蛋白 单纯疱疹病毒 疫苗 

分 类 号：Q78[生物学—分子生物学] R392[医药卫生—免疫学]