[The 38,000 protein of Mycobacterium tuberculosis is overexpressed in Escherichia coli].
OBJECTIVE
To obtain recombinant 38,000 protein in large quantities and to study its immunologic characteristics by stable expression of the gene encoding for 38,000 antigen of Mycobacterium tuberculosis in E. coli.


METHODS
Expression plasmid was constructed with DNA recombinant technique. Positive clones were screened using double digestion and polymerase chain reaction. Recombinant plasmid was transformed into E. coli. Then E. coli carrying recombinant plasmid were induced. The expression of 38,000 antigen was identified by SDS-polyacrylamid gel electrophoresis (PAGE) and immunoblotting. Stained gel was scanned to detect expression level of recombinant antigen.


RESULTS
Gel stained with coomassie blue G-250 showed that the induced E. coli carrying recombinant plasmid can produce 38,000 protein at high level. Gel scan showed that 38,000 antigen expression in E. coli was about 36% - 40% of total cellular protein. The recombinant 38,000 antigen existed mostly in inclusion bodies. The recombinant antigen can react with antibodies: serum of pulmonary tuberculosis patients and the goats immuned with Mycobacterium tuberculosis.


CONCLUSIONS
Constructed recombinant E. coli can overproduce 38,000 antigen of Mycobacterium tuberculosis. Inclusion bodies are easy to purify and can protect the recombinant antigen from protease, on the other hand, it has not biological activity unless the denatured protein is accurately folded.
He
X
X
Tuberculosis Research Laboratory, The 309th Hospital of PLA, Beijing 100091.
Zhuang
Y
Y
Zhang
X
X
Xiong
Z
Z
Chang
Z
Z
D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics*; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis*
D000942:Antigens, Bacterial; D011994:Recombinant Proteins