[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.

Conclusion: The E. coli.-BCG shuttle vector is constructed successfully which could express exogenous antigen gene as a chimeric exported membrane.