Construction and identification of a prokaryotic expression plasmid encoding HPV16E7-HSP70 fusion gene
- VernacularTitle:HPV16E7-HSP70融合基因原核表达质粒的构建和鉴定
- Author:
Shuwei ZHAO
;
Jie QIU
;
Xinjiang YING
;
Qing YE
;
Aihua SUN
- Publication Type:Journal Article
- Keywords:
Papillomavirus, Human;
Heat- Shock Protein 70;
Recombinant Fusion Proteins;
Plasmids
- From:
Chinese Archives of Otolaryngology-Head and Neck Surgery
2006;0(02):-
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE To construct a prokaryotic expression plasmid encoding HPV16E7-HSP70 fusion gene for further study on the immunity of HPV16E7- HSP70 fusion protein against laryngeal carcinoma. METHODS HPV16E7 was PCR-amplified,digested by NheI and SacI,and ligated into pET28a. HSP70 was cloned into pGEMTeasy,then recut from the vector by SalI and NotI and ligated into pET28a-HPV16E7. PCR amplification, restrict enzyme digestion, DNA sequencing, IPTG induction and Western Blot were used to identify the recombinant plasmid. RESULTS Double digestion and PCR amplification of the recombinant plas- mid have shown that the size of the inserted fragment is as expected. Sequence analysis has demonstrated that the inserted fragment encodes for the HPV16E7- HSP70 fusion gene. IPTG induction and Western Blot have shown that the fusion protein is expressed suc- cessfully in the prokaryotic expression plasmid. CONCLUSION The recombinant prokaryotic expression plasmid pET28a-HPV16E7-HSP70 has been con- structed successfully.
