[The construction and expression of Rv3872 as a member of virulent protein secret system from Mycobacterium tuberculosis in Bacillus Calmette-Guerin].
OBJECTIVE
To construct, express and identify a recombinant prokaryotic expression vector pET-3872 carrying Rv3872 of Mycobacterium tuberculosis H37Rv strain, and to construct the recombinant Bacillus Calmette-Guerin (rBCG) strain expressing Rv3872 (PE35)of Mycobacterium tuberculosis H37Rv strain.


METHODS
The Rv3872 gene was amplified by PCR from Mycobacterium tuberculosis H37Rv strain and cloned into prokaryotic expression vector pET32a(+). The recombinant plasmid pET-3872 was sequenced and transformed into E.coli BL21(DE3), and was induced with IPTG to express a 35 kD fusion protein, which was confirmed as His-Rv3872 by Western blot. The expression product was purified and the new Zealand rabbits were immunized. Rv3872 was amplified by PCR and cloned into E.coli-Mycobacteria shuttle vector pMV361. The recombinant vector was named as pMV-3872, and then pMV-3872 was transformed the to BCG via electroporation, the recombinant BCG-3872 strain. The PE35 protein expression of BCG-3872 was induced with heat shock reaction. Then BCG-3872 culture supernatant and bacterial precipitation were collected respectively and analyzed by Western blot.


RESULTS
A recombinant fused expression vector pET-3872 was constructed and His-3872 protein was confirmed by Western blot. BCG-3872 strain was constructed and Western blot confirmed the presenle of the PE35 protein in the supernatant of BCG-3872 strain culture.


CONCLUSIONS
The prokaryotic expression vector pET-3872 was constructed, and the 35 kD fusion protein His-3872 was expressed and purified successfully, which provides a tool for further functional study of the Rv3872 . The recombinant BCG strain expressing PE35 protein, BCG-3872 strain, was constructed successfully, which facilitates further study on BCG-3872 immune function.
Shi
Xin-xin
XX
Department of Infection and Immunity, Preclinical and Forensic Medicine School of West China Medical Center, Sichuan University, Chengdu 610041, China.
Bao
Lang
L
Qiu
Yun-qing
YQ
Guo
Si
S
D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D015153:Blotting, Western; D005822:Genetic Vectors / Q000235:genetics; D009163:Mycobacterium bovis / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction
D000942:Antigens, Bacterial