人乳头瘤病毒6b型早期蛋白E6和E7基因的克隆及表达

目的:从尖锐湿疣(CA)标本中克隆出人乳头瘤病毒6b型(HPV-6b)早期蛋白E6、E7基因,并进行序列分析比对及原核表达,为HPV感染的检测和基因工程疫苗研究奠定基础。方法:用PCR法,从CA标本中扩增HPV-6的E6、E7基因,构建pUCHPV-6E6、pUCHPV-6E7两个重组体,酶切鉴定及测序分析比对。经双酶切与表达载体pGEX-5X-1定向连接,构建pGEX-5X-l重组表达质粒,转入BL21大肠杆菌,IPTG诱导GST融合蛋白表达及Westernblot鉴定。结果:克隆出HPV-6b早期蛋白E6、E7基因,成功构建pUCmHPV-6bE6、pUCmHPV-6bE7重组质粒,克隆获得HPV-6bE6、E7基因与GenBank标准株序列完全相同,经双酶切定向克隆,成功构建重组表达质粒pGEX-5X-lHPV-6bE6和pGEX-5X-lHPV-6bE7,转入BL21大肠杆菌,高效表达GST融合蛋白。结论:本研究克隆出的HPV-6bE6、E7基因与标准株相同,HPV-6bE6、E7GST融合蛋白获得高效表达,为HPV-6bE6、E7基因表达产物的纯化、体外活性以及E6、E7为靶位的基因疫苗研究奠定基础。

Molecular cloning and expression of E6 and E7 genes of human papillomavirus type 6b

Objective: To provide target antigens for the development of condyloma acuminatum (CA) therapeutic vaccine against HPV infection. Methods: HPV DNA was extracted from samples of CA and HPV-6b E6, E7 genes were amplified by PCR. The recombinant plasmids were constructed by cloning the gene fragment into plasmid pUCm-T. The recombinant plasmids were identified by restriction enzyme analysis and DNA sequencing. Target DNA fragments in recombinant pUCms were digested with EcoR I and Sal I restriction enzymes, and subcloned into expression vector pGEX-5X-1 induced by IPTG for their recombinant protein expressions. Twenty-six glutathione S-transferase (GST) fusion proteins, expressed in E. coli BL21, were characterized by 10% SDS-PAGE and Western blot. Results: The E6 and E7 genes of HPV-6b were cloned by PCR successfully. The recombinant plasmids pUCm/HPV-6bE6 and pUCm/HPV-6bE7 were constructed and confirmed with enzyme digestion and sequencing. The recombinant expression plasmids of pGEX-5X-1/HPV-6bE6 and pGEX-5X-1/HPV-6bE7 were obtained. In E. coli BL21, GST fusion proteins of HPV-6b E6 and E7 were stably expressed about 41kD and 37kD, respectively. Conclusion: Both types of recombinant plasmids which contain HPV-6b E6, E7 genes have been successfully constructed. The recombinant expression plasmids of HPV-6bE6 and E7 can stably express GST fusion proteins. These results can serve as a base for further studies on the usefulness of the genes and their expression products in the development of new therapeutic vaccine against HPV infecton.