[Molecular cloning and expression of the immunodominant protein Ag85A from Mycobacterium tuberculosis H37Rv strain].
OBJECTIVE
To provide the target gene and target antigen for the development of new vaccine against tuberculosis.


METHODS
According to the gene sequence encoding protein Ag85A from Mycobacterium tuberculosis H37Rv strain, we designed a pair of oligonucleotide primers, obtained the gene by using polymerase chain reaction, and inserted the gene into the BamH I and EcoR I site of plasmid pBK-CMV to construct recombinant plasmid, and after that, the recombinant plasmid was transferred into E. coli XL1-Blue MRF' and induced with IPTG. The expression product of the gene was analyzed by using SDS-PAGE and western-blotting.


RESULTS
The gene encoding the protein Ag85A of Mycobacterium tuberculosis H37Rv strain was successfully amplified by using PCR. A recombinant shuttle plasmid was constructed. The recombinant plasmid stably expressed recombinant Ag85A protein relative molecular mass 32 x 10(3) in E. coli XL1-Blue MRF'.


CONCLUSIONS
A recombinant plasmid which contains the gene encoding the protein Ag85A of Mycobacterium tuberculosis H37Rv strain has been successfully constructed. The recombinant plasmid can stably express recombinant protein relative molecular mass 32 x 10(3) in E. coli XL1-Blue MRF'. These results could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of new vaccine against tuberculosis.
Xie
Yongen
Y
Research Unit of Infection and Immunity, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Bao
Lang
L
Hu
Changhua
C
Zhang
Wanjiang
W
Chen
Wei
W
D000217:Acyltransferases / Q000096:biosynthesis / Q000235:genetics*; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018014:Gene Transfer Techniques; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics / Q000276:immunology*; D010957:Plasmids / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D032581:Tuberculosis Vaccines / Q000276:immunology
D000942:Antigens, Bacterial; D011994:Recombinant Proteins; D032581:Tuberculosis Vaccines; D000217:Acyltransferases; C112580:antigen 85A, Mycobacterium tuberculosis