Expression Of The MPB64 Protein Of Mycobacterium Bovis In Escherichi Coli And Preparation Of Monoclonal Antibodies Against MPB64

Mycobacterium is a Gram-positive bacteria, and it is red in the Ziehl-Neelsen stain. Most strains grow slowly in the environment. Some strains can cause tuberculosis or tuberculosis-like disease of human and animals, and these strains composed to Mycobacterium tuberculosis complex (MBTC). MPB64 protein is an early filtrate protein which is secreted by the members of MBTC. Now it is usful in the identification of strains and the research of the Mycobacterium vaccines.To preparate the monoclonal antibodies of MPB64 protein, the whole gene fragments including initiation codon of mpb64 was amplified by PCR from the genome DNA of Mycobacterium bovis Vallee III, then inserted into the pET32a(+) to construct prokaryotic expression vector, named pET-mpb64. Recombinant fusion protein rMPB64 was expressed in pET-mpb64-transformed Escherichi coli cells after induction with IPTG, as demonstrated by SDS-PAGE analysis. The recombinant protein could be recognized by TB antisera of cattle in the western blotting. Meanwhile the same whole genes including initiation codon was insert into eukaryotic expression vector pcDNA3.1 (+) to construct eukaryotic expression vector pc-mpb64, which caused express of mpb64 gene in mammalian cells by transfected into SP2/0 cell. BALB/c mice were immunized with pc-mpb64, then the spleen cells were fused with SP2/0 cells with PEG3250 in 5 days after the last boosting immunizing. Three monoclonal antibodies (A2E5,B3G8,C5F10) about MPB64 protein were obtained after three to four times subcloning. The A2E5 was identified to be IgM subtype and lambda light chain. B3G8 and C5F10 were identified to be IgG2a subtypes and kappa light chain. All three McAbs can react with recombinant fusion protein and PPD in ELISA experiment, but not with rMPB70-83-ESAT6, Moreover all three McAbs could not react with these proteins in western blotting. So that they might recognize structural epitope of the protein.In summary, recombinant MPB64 protein of Mycobacterium bovis was expressed in E. coli and three hybridoma cell lines secreting McAbs directed against MPB64 protein were developed, which can be used to study about structure and function of MPB64 protein, and to establish the detection assay about MPB64 in early filtrate protein.