Expression,identification and purification of the resuscitation-promoting factor A of mycobacterium tuberculosis
- VernacularTitle:结核分枝杆菌RpfA蛋白的原核表达、鉴定和纯化
- Author:
Lei XU
- Publication Type:Journal Article
- Keywords:
Mycobacterium tuberculosis;
RpfA;
Clone;
Expession;
Purification
- From:
Journal of Chongqing Medical University
2003;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct the Resuscitation-promoting factor A(RpfA) expression plasmid of Mycobacterium tuberculosis, and express and purify it in E.coli.Methods:RpfAgene of Mycobaeterium 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 His-tag in-gel Stain and purified by using affinity chromatograghy.Results:The size of the RpfA gene digested by restricted enzymes accorded with the theoretical size and the DNA sequence with the reported one in literatures.66ku protein was found by SDS-PAGE and His-tag in-gel Stain.Conclusion:The recombinant expression plasmid pET32a(+)-RpfA was successfully cloned and constructed, and the recombinant protein with high purity was obteined;which laid the basis for further study.
