人乳头瘤病毒11型E7蛋白基因的克隆和表达  

Cloning and Expression E7 Protein Gene of Human Papillomavirus Type 11 in E. Coli

作　　者：王飞[1] 毕志刚[1] 王群[2] 李光富[3] 王新军[3] 张兆松[3] 

机构地区：[1]南京医科大学第一附属医院皮肤科,210029 [2]广东省人民医院皮肤科 [3]南京医科大学分子生物研究所

出　　处：《中华皮肤科杂志》2004年第7期416-418,共3页Chinese Journal of Dermatology

基　　金：江苏省重点学科基金(135-3)

摘　　要：目的从尖锐湿疣皮损标本中扩增出人乳头瘤病毒11型(HPV11)E7蛋白基因,并进行表达、纯化和鉴定。方法用PCR法,从尖锐湿疣标本中扩增出HPV11E7蛋白基因,与pGEX-6P-1载体连接成重组表达质粒pGEX-6P-1/E7;重组质粒转入BL21大肠杆菌,经诱导表达融合蛋白GST-E7;该蛋白经剪刀酶切除GST,GlutathioneSepharose4B亲和层析柱纯化,SDS-PAGE和蛋白质印迹法检测鉴定。结果经酶切及序列分析鉴定,重组质粒pGEX-6P-1/E7成功构建,诱导后高表达GST-E7融合蛋白并得到纯化,蛋白质印迹证实表达蛋白为E7蛋白。结论获得高表达、高纯度的HPV11E7蛋白,为该蛋白的功能及免疫学分析及尖锐湿疣疫苗研究打下了基础。Objective To clone and express the gene encoding E7 protein of human papillomavirus type 11 (HPV11). Methods The gene encoding for E7 protein of HPV11 was amplified by PCR, cloned into vector PGEX-6P-1 to form PGEX-6P-1/E7 plasmid, and then transfected into the E. coli BL21. The GST-E7 fusion protein was expressed when induced by adding IPTG and purified with glutathione sepharose 4B affinity column. The GST portion of GST-E7 fusion protein was cleaved by thrombin. SDS-PAGE and Western blotting were used to detect the protein expression. Results The recombinant plasmid was identified and confirmed with enzyme digestion and sequencing. A high level expression of GST-E7 fusion protein was obtained and purified successfully. SDS-PAGE and Western blotting suggested that the E7 was a 11 000 molecular weight protein reacting with anti-E7 antibody. Conclusions Highly expressed E7 protein of HPV11 with high purity is obtained, which might be used for the study of its function, immunological analysis, and the study of the vaccine of genital warts.

关 键 词：人乳头瘤病毒11型 E7蛋白基因 基因克隆 基因表达 人乳头瘤病毒感染 尖锐湿疣 

分 类 号：R346[医药卫生—基础医学]