Expression,purification and characterization of recombinant multiepitope peptide of ESAT-6 of Mycobacterium tuberculosis
- VernacularTitle:结核分枝杆菌ESAT-6蛋白免疫优势肽段的重组表达、纯化与鉴定
- Author:
Haibo WANG
;
Zhongyuan ZHU
;
Xianjie LIU
- Publication Type:Journal Article
- Keywords:
Mycobacterium tuberculosis;
ESAT-6 protein;
epitope;
expression
- From:
Chinese Journal of Clinical Laboratory Science
2006;0(03):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To obtain sufficient recombinant multiepitope peptide of ESAT-6.Methods DNA encoding ESAT-6 peptide was amplified by PCR using a pair of primers which were designed in accordance with the reported ESAT-6 gene sequence.The ESAT-6P gene was inserted into the prokaryotic expression vector pET-30a with the N-terminal 6His-tag.The recombinant peptide vector pET30a-ESAT-6P was transformed into E.coli BL21(DE3) via chemical transformation.The positive clone was screened by means of PCR.The target peptide was expressed in E.coli after induction with IPTG and analyzed by SDS-PAGE.The immunogenicity of the peptide was evaluated by dot immunobinding assay.Results The target peptide was successfully expressed and purified.The solubility analysis showed that the recombinant peptide existed as inclusion body in the E.coli.DIBA indicated that the ESAT-6P was specifically reactive to sera from TB patients.Conclusions The recombinant multiepitope peptide of ESAT-6 engineering bacteria has been obtained,which will be quite helpful to develop novel specific diagnostic reagents and effective vaccine.
