[Mycobacterium tuberculosis secreting protein Ag85B-ESAT6 fused expression and purification].
OBJECTIVE
To investigate the fused expression of secreted protein Ag85B-ESAT6 of Mycobacterium tuberculosis, and to provide a promising preventive subunit vaccine against tuberculosis.


METHODS
The gene encoding Ag85B and ESAT6 protein was amplified by PCR from genome of Mycobacterium tuberculosis H(37)Rv strain, and inserted into cloning vector P(GEM)-T-easy. After sequence analysis, and digestion by restriction endonuclease, Ag85B-ESAT6 was cloned into corresponding sites of the expression vector P(PRO) EXHT, and the recombinant plasmid was transformed into expressive strain E. coli DH5 alpha, induced with IPTG and fusion protein was purified by Ni-NTA purification system. The specific antibody titer in the sera of BALB/c mouse immunized with two fusion protein was detected by ELISA.


RESULTS
The sequences of Ag85B and ESAT6 by PCR amplification were identical to those reported by GenBank. The recombinant plasmid fused expression protein of Ag85B-ESAT6 with relative molecular mass (Mr) of 37,000, which was confirmed by Western-blot analysis with specific monoclonal antibody against 6 x HismAb. The fused expression protein was insoluble. It could be purified by Ni-NTA purification system. The specific antibody titer in the sera of BALB/c mouse immunized with fusion Ag85B-ESAT6 was 1:1000 and that of mouse immunized with fusion protein ESAT6-Ag85B was 1:5000.


CONCLUSIONS
Secreted protein Ag85B-ESAT6 of Mycobacterium tuberculosis was successfully fused expressed in E. Coli DH5 alpha. It may become a new type of vaccine against tuberculosis.
Department of Microbiology, Faculty of Preclinical Medicine, Fourth Military Medical University, Xi'an 710033, China.
Chang-hong
CH
Shi
Xiong-lin
XL
Fan
Zhi-kai
ZK
Xu
Yuan
Y
Li
Yin-lan
YL
Bai
Ying
Y
Xue
D000217:Acyltransferases / Q000235:genetics* / Q000276:immunology; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D007371:Interferon-gamma / Q000096:biosynthesis; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D011993:Recombinant Fusion Proteins; D007371:Interferon-gamma; D000217:Acyltransferases; C121433:antigen 85B, Mycobacterium tuberculosis