Expression,identification and purification of the resuscitation-promoting factor B of Mycobacterium tuberculosis
- VernacularTitle:结核分枝杆菌复活促进因子B的原核表达、鉴定和纯化
- Author:
Lei XU
- Publication Type:Journal Article
- Keywords:
Mycobacterium tuberculosis;
RpfB;
Clone;
Expession;
Purification
- From:
Journal of Chongqing Medical University
1986;0(03):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct the Resuscitation-promoting factor B(RpfB)expression plasmid of Mycobacterium tuberculosis, and express and purify it in E.coli.Methods:RpfB gene of Mycobacterium tuberculosis was amplified with PCR method from the H37Rv,cloned into pET32a(+)vector,and then transformed into E.coli Top10.After identifying by restriction digestion and DNA sequencing,the constructed recombinant plasmid was transformed into E.coli BL21,and expressed the recombinant protein under the control initiated by T7 after induction with IPTG.The target protein was identified by Western-blot and purified by using affinity chromatograghy.Results:The size of the RpfB gene digestedby restricted enzymes accorded with the theoretical size and the DNA sequence with the reported one in literatures.61KDa protein was found by SDS-PAGE and Western-blot.Conclusion:The recombinant expression plasmid pET32a(+)-RpfBv of RpfB gene was successfully cloned and constructed,and the recombinant protein with high purity was obteined;which laid the basis for further study.
