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  • Title: [Constructing shuttle plasmid fusion expressing Ag85B-ESAT-6 on the surface of Mycobacterium].
    Author: Shi CH, Fan XL, Shi JR, Xu ZK, Li Y, Bai YN, Xun Y.
    Journal: Zhonghua Jie He He Hu Xi Za Zhi; 2004 Apr; 27(4):249-52. PubMed ID: 15144616.
    Abstract:
    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.
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