Cloning and Expression of HPV18 E6 Gene
10.3969/j.issn.0253-9896.2009.12.001
- VernacularTitle:HPV18E6基因的原核克隆及表达
- Author:
Hairong JIANG
;
Fangyi PENG
;
Yuanxiang CHEN
;
Shengzhen CHEN
;
Zhihua LIN
;
Fangliang PENG
;
Weibing ZHAO
- Publication Type:Journal Article
- Keywords:
laryngeal neoplasms carcinoma gene expression prokaryotic cells electrophoresis;
polyacrylamide gel blotting;
Western
- From:
Tianjin Medical Journal
2009;37(12):993-995
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To express the protein of HPV18E6 based on pET-32a(+) at high level and study the expression and significance of HPV18E6 proteins in laryngeal carcinoma. Methods: The HPV18E6 gene was amplified by PCR and cloned into pET-32a(+). The amplified fragment was inserted into the plasmid pET32a (+) that was digested with BamHⅠand Hind Ⅲ. The recombinant plasmid pET32/E6 was transformed into E.coli JM109 which was selected with ampicillin. The recombinant plasmids were successfully introduced into E.coli BL21(DE 3) and were induced by IPTG. SDS-PAGE and Western blot analysis were used to detect the confusion protein. Finally, the optimization of expression conditions, such as temperature, concentration of IPTG, was studied. Results: The recombinant plasmids were identified and confirmed with enzyme digestion and sequencing. The BL21(DE3) transformed recombinant plasmid pET32/E6 had expressed HPV18E6 recombinant protein effectively. The optimum conditions of expression were 37 ℃, 1 mmol/L IPTG. Conclusion:Prokaryotic expression vector pET-32a(+)-HPV18E6 was successfully constructed. The high-level expression of HPV18E6 was achieved in E.coli BL21(DE3).
