[Molecular cloning, purification, and serological characterization of four specific antigens of Mycobacterium tuberculosis].
OBJECTIVE
To express the 14,000, 37,000, and 6,000 early secretory antigenic target (ESAT-6) and mtb81 antigen genes in bacteria, and to purify the product and determine their activity.


METHODS
The 14,000, 37,000 , ESAT-6, and mtb81 antigen genes were amplified from Mycobacterium tuberculosis genomic DNA by polymerase chain reactions and cloned into pGEX 4T-1 expression vector. BL21 strain of Escherichia coli was transformed with the recombinant vectors and induced to express recombinant proteins. The proteins were purified by affinity chromatography. The biological activity of purified proteins were estimated by enzyme-linked immunoabsorbant assay (ELISA).


RESULTS
The BL21 strains of Escherichia coli with recombinant vectors showed high level of 14,000, 37,000, ESAT-6, and mtb81 gene expressions after induction. The products were purified successfully and showed high antigenicity and specificity. The sensitivity of 38,000, 14,000, ESAT-6, and mtb81 were 54%, 60%, 44%, and 36%, respectively.


CONCLUSIONS
The expressions and purifications of recombinant 14,000, 37,000, ESAT-6, and mtb81 antigens with natural activity facilitate their research and application.
Xu
Lei
L
Department of Biochemistry, Medical College, Tongji University, Shanghai 200092, China.
Li
Hui-yun
HY
Ma
Li-heng
LH
D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014376:Tuberculosis / Q000097:blood / Q000276:immunology
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011994:Recombinant Proteins