Prokaryotic expression and polyclonal antibody preparation of nonstructural protein 2 transactivated protein of hepatitis C virus
10.3760/cma.j.issn.1000-6680.2009.04.006
- VernacularTitle:丙型肝炎病毒非结构蛋白2反式激活蛋白的原核表达及多克隆抗体制备
- Author:
Yuan HONG
;
Mengdong LAN
;
Qi WANG
;
Liying ZHANG
;
Xiucai LIU
;
Xiaoguang LI
;
Xiaohua HAO
;
Jun CHENG
- Publication Type:Journal Article
- Keywords:
Trans-activators;
Gene expression;
Antibodies;
Immunohistochemistry;
Hepacivirus
- From:
Chinese Journal of Infectious Diseases
2009;27(4):217-220
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express nonstructural protein 2 transactivated protein (NS2TP) of hepatitis C virus (HCV) in the prokaryotic expression system and prepare polyclonal antibody,and to study the expressions in different liver tissues.Methods NS2TP gene was amplified by polymerase chain reaction (PCR) technique and cloned into the prokaryotic expression vector pET-32a(+),which was transformed into E.coli BL21.The protein was induced by isopropyl thiogalactose (IPTG) and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blotting assay.The recombinant protein were expressed and purified in a large amount.The rabbit was immunized with the purified protein to prepare polyelonal antibody.The liver tissues of patients with chronic HCV infection and healthy controls were detected by immunohistochemistry method.Results The recombinant NS2TP protein (relative molecular mass:33×103 ) and polyclonal antibody with high titer and specificity were successfully prepared.NS2TP expressions in the liver of patients with chronic HCV infection were higher than those of healthy controls,and were mainly distributed in the nucleus of hepatocytes.Conclusions The NS2TP expression level and intracellular location in liver tissue of patients with chronic HCV infection are understanded,which could bring new clues for further study of the biological function of NS2TP and the pathogenesis of HCV infection.
