HPV16地方分离株E6基因的克隆与原核表达研究  

Cloning and expression in Escherichia coli of E6 gene from human papillomavirus type 16 isolate.

作　　者：商庆龙[1] 刘雪梅[2] 王燕[1] 李承刚[1] 邵迪[1] 陈思佳[1] 韩聪[1] 谷鸿喜[1] 

机构地区：[1]哈尔滨医科大学微生物教研室,150081 [2]哈尔滨市道里区疾病预防控制中心

出　　处：《中国地方病学杂志》2007年第3期280-282,共3页Chinese Jouranl of Endemiology

基　　金：黑龙江省科技攻关项目（CB02C111）;哈尔滨市科技攻关计划项目（2004AA3CS176-1）

摘　　要：目的克隆人乳头瘤病毒（HPV16E6）基因并在大肠埃希菌中表达，对表达产物进行鉴定。方法用PCR方法从克隆质粒pUC19-HPV16E6E7中扩增HPV16E6基因，采用定向克隆构建pQE30-HPV16E6原核表达质粒．利用酶切和序列测定鉴定重组质粒。将pQE30-HPV16E6转化大肠埃希菌BL21（DE3），建立重组工程菌pQE30-HPV16E6／BL21（DE3）。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导，SDS—PAGE分析蛋白表达情况，利用Western blot鉴定抗原特异性。结果PCR产物470bp，重组质粒经酶切和序列测定证实构建正确。SDS—PAGE分析在18×10^3处有蛋白条带出现，与预期一致。Western blot分析证实目的条带与HPV16E6抗体有特异性反应。结论成功构建了HPV16E6基因的基因工程菌株，能高效表达E6蛋白，表达蛋白具有良好的免疫反应性。Objective To construct and identify gene engineering bacteria strain and to produce HPV16 E6 protein. Methods HPV16E6 coding region from the second ATG was amplified from pUC-HPV16E6E7 by high-fidelity PCR. The amplified products were cloned into plasmid pQE30 and sequenced. Subsequently, pQE30- HPV16E6 was constructed by inserting the HPV16E6 gene into pQE30. The E6 protein was expressed in the host strain BL21 （DE3） after IPTG induction, and identified by SDS-PAGE and Western blot analysis. Results The PCR product was 470 bp. The recombinant plasmid pQE30-HPV16E6 was confirmed by restriction digestion and sequencing. SDS-PAGE showed that the gene engineering bacteria pQE30-HPV16E6/BL21 （DE3） expressed proteins with molecular weight of 18 × 10^3 which existed in inclusion bodies. Western blot validated the specificity of the target protein for the HPV16E6 polyclonal antibody. Conclusions HPV16E6 gene was cloned and expressed in bacteria successfully, and the recombinant protein was specifically identified by Western blot.

关 键 词：乳头瘤病毒 人 基因表达 大肠杆菌 

分 类 号：R686[医药卫生—骨科学]