[Constructing shuttle plasmid fusion expressing Ag85B-ESAT-6 on the surface of Mycobacterium].
OBJECTIVE
To construct the E. coli.-BCG (Bacille Calmette-Guerin) shuttle vector expressing Mycobacterium tuberculosis secreted protein Ag85B-ESAT-6 on the surface of Mycobacterium vaccae.


METHODS
The gene fragment containing 19 000 antigen (19-ss) were amplified by polymerase chain reaction (PCR) from the Mycobacterium tuberculosis H(37)Ra. We cloned the 19ss gene into the E. coli.-BCG shuttle vector pOLYG and named the pCW, which can shuttle and express exogenous antigen gene on cell wall of Mycobacterium. Then Mycobacterium tuberculosis secret protein Ag85B and ESAT-6 gene were cloned into the vector and determined by indirect immunofluorescence.


RESULTS
The sequence of 19-ss gene was identified with Genbank reported by sequencing. The constructed E. coli.-BCG shuttle vector using 19ss gene had the function of shuttle between E. coli. and Mycobacteria. By indirect immunofluorescence technique the secreted protein Ag85B-ESAT-6 can be fused and expressed on surface of Mycobacterium vaccae.


CONCLUSIONS
The E. coli.-BCG shuttle vector is constructed successfully which could express exogenous antigen gene as a chimeric exported membrane.
Shi
Chang-hong
CH
Department of Microbiology, Fourth Military Medical University, Xi'an 710033, China. changhongshi@163.net
Fan
Xiong-lin
XL
Shi
Jie-ran
JR
Xu
Zhi-kai
ZK
Li
Yuan
Y
Bai
Yin-nan
YN
Xun
Ying
Y
D000217:Acyltransferases / Q000096:biosynthesis* / Q000235:genetics; D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D001500:BCG Vaccine; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D009161:Mycobacterium / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D019444:Vaccines, DNA
D000942:Antigens, Bacterial; D001500:BCG Vaccine; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; D000217:Acyltransferases; C121433:antigen 85B, Mycobacterium tuberculosis