Construction of HPV18E7 recombinant plasmid and exploration of its optimization expression condition in Escherichia coli
10.3969/j.issn.1671-8348.2013.30.022
- VernacularTitle:HPV18E7重组质粒的构建及在大肠杆菌中的表达优化
- Author:
Renjian HU
;
Jiali CAI
;
Li LIU
;
Manyu TU
;
Tao XU
;
Cuirong DU
;
Jia LUO
;
Sen DING
- Publication Type:Journal Article
- Keywords:
human papillomavirus type 18;
HPV18E7 gene;
HeLa cell line;
auto-induction medium
- From:
Chongqing Medicine
2013;(30):3647-3649
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct recombinant plasmids containing HPV18E7 gene ,and explore the optimization condition of its expression in Escherichia coli .Methods The genomic DNA extracted from HeLa cell line which served as a template to the HPV18 E7 gene was amplified using PCR method ;and the amplified product of HPV18E7 gene was connected to the pET-32a(+ ) vector ,which composed the pET-32a(+ )-HPV18E7 recombinant plasmid ;the positive recombinant plasmids were transformed into BL21-DE3-pLysS competent cells and the optimized expression condition was explored in order to obtain a large amount of HPV18E7 oncogenic protein .Results The fragment length of PCR products of HeLa cell genomic DNA was consistent with that of HPV18 E7 gene .In LB medium ,the expression level of the target protein was not high under such conditions as different concentra-tion of IPTG and lactose ,different temperatures and different induction starting amount .Therefore the ZYM-5052 auto-induction medium was tried in this experiment ,and the expression amount of the fusion protein was much higher than that induced with IPTG and lactose .Conclusion The amount of HPV18E7 fusion protein in ZYM-5052 automatic induction medium is much higher than that induced with IPTG and lactose .
