人乳头瘤病毒11型E6的原核表达  

Expression of E6 Protein Gene of Human Papillomavirus Type 11 in E.Coli

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

机构地区：[1]南京医科大学第一附属医院皮肤科,江苏南京210029 [2]南京医科大学分子生物研究所,江苏南京210029 [3]广东省人民医院皮肤科,广东广州510080

出　　处：《中国皮肤性病学杂志》2004年第5期257-259,268,共4页The Chinese Journal of Dermatovenereology

基　　金：江苏省重点学科基金 (1 35 - 0 3)

摘　　要：目的　构建、克隆人乳头瘤病毒 11型 (HPV11)E6蛋白基因 ,并进行表达、纯化和鉴定。方法　用PCR法 ,从尖锐湿疣 (CA)标本中扩增出HPV 11型E6蛋白基因 ,与 pET3 2a载体连接成重组表达质粒pET3 2a/E6;重组质粒转入BL2 1大肠杆菌 ,经诱导表达硫氧还蛋白E6融合蛋白 (Trx E6) ;该蛋白经 3SNTAResin纯化柱纯化 ,SDS PAGE和WesternBlotting检测鉴定。 结果　经酶切及序列分析鉴定 ,重组质粒 pET3 2a/E6成功构建 ,诱导后高表达Trx E6融合蛋白 ,WesternBlotting鉴定证实表达蛋白分子量为 3 60 0 0D的Trx E6融合蛋白。结论　获得高表达、高纯度的HPV11E6蛋白 ,为该蛋白的功能及免疫学分析及CA的疫苗研究打下了基础。Objective To express the gene encoding E6 protein of Human Papillomavirus type 11(HPV11) and purify the E6 protein.Methods The HPV11 E6 gene was amplified from samples of condylome acumiuatum(CA) by PCR,cloned into vector pET32a to form pET32a/E6 plasmid and then transfected into the E.coli BL21.The thioredoxin-E6 fusion protein(Trx-E6) was expressed when induced by adding of IPTG and purified with 3S NTA Resin affinity column.SDS-PAGE and Western blotting were used to detect the expressed protein. Results The recombinant plasmid was identified and confirmed with enzyme digestion and sequencing.A high level expression of Trx-E6 fusion protein was obtained and purified successfully.SDS-PAGE and Western Blotting suggested that the recombinant fusion protein was a 36,000 Dalton molecular weight protein reacted with anti-His antibody.Conclusion Trx-E6 protein of HPV11 could be expressed with high efficiency in prokaryotic expression system.

关 键 词：人乳头瘤病毒11型 E6蛋白基因 检测 基因克隆 基因表达 尖锐湿疣 

分 类 号：R373.9[医药卫生—病原生物学]