[人乳头瘤病毒16型L1蛋白的原核表达及表达条件优化]

[Prokaryotic expression of HPV 16L1 and optimization of expression conditions].

作者信息

Peng Fang-yi, Jiang Hai-rong, Chen Yuan-xiang, Chen Sheng-zhen, Lin Zhi-hua, Peng Fang-liang, Zhao Wei-bin, Chen Bao-de

机构信息

School of Pharmacy and Bioengineering, Chongqing University of Technology, and Department of Gynecology and Obstetrics, Chongqing Forth People's Hospital, Chongqing 400050, China.

出版信息

Zhejiang Da Xue Xue Bao Yi Xue Ban. 2010 Jul;39(4):395-8. doi: 10.3785/j.issn.1008-9292.2010.04.010.

DOI:10.3785/j.issn.1008-9292.2010.04.010

PMID:20731039

Abstract

OBJECTIVE

To construct the HPV16 L1 prokaryotic expression plasmid and to optimize its expression.

METHODS

A pair of primers was designed according to plasmid sequences of pGEX-KG and the HPV16L1 genes published by GeneBank. The DNA fragment of 1500 bp was amplified by PCR from the HPV recombinant plasmid with HPV16L1 gene, then cloned into pGEX-KG and transformed into the host E.coli strain JM109. The pGEX-KG-HPV16L1 plasmid was taken and transformed into BL21(DE3) for expression. Induced by IPTG at 37 degree, the expression product of HPV16L1 gene was identified by SDS-PAGE and Western blot.

RESULTS

HPV16L1 fusion protein was expressed successfully in the form of inclusion bodies. The molecular weight was 83 kD. Meanwhile, the optimum condition of HPV16L1 fusion protein expression was induced with 1.0 mmol*L(-1) IPTG for 4 h. The fusion protein reacted specifically with antibodies against HPV16L1.

CONCLUSION

The prokaryotic expression vector of HPV16L1 gene has been constructed and expressed in E.coli successfully.

摘要

目的

构建人乳头瘤病毒16型（HPV16）L1蛋白原核表达质粒并对其表达进行优化。

方法

根据GenBank公布的pGEX-KG质粒序列及HPV16 L1基因设计一对引物，以含HPV16 L1基因的HPV重组质粒为模板，通过PCR扩增出1500 bp的DNA片段，克隆至pGEX-KG载体，转化宿主菌大肠杆菌JM109。提取pGEX-KG-HPV16L1质粒，转化至BL21(DE3)进行表达。37℃用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western blot）鉴定HPV16 L1基因的表达产物。