Expression Of Mycobacterium Tuberculosis HSP65 And Its Immunological Properities

Mycobacterium tuberculosis (MTB) Heat Shock Protein 65(HSP65)which belongs to HSP60s,encoding by gene groEL2 ,is a kind of highly conservative protein. It is one of the important protective antigens for resistance of MTB infection. The protein has strong immunogenicity which has been proved that about 20% reactive T cell can discriminate HSP65 in MTB infected mouse, thus HSP65 may a key point for precaution and therapy of tuberculosis (TB).This research used the molecular biology method to construct prokaryotic recombinant plasmid and eukaryotic recombinant plasmid based on HSP65 of MTB .Compared with BCG, to evaluate the immunological properties and protective efficacy of them against MTB infection, in order to find a new efficient vaccine against TB. The results as follows:1. The gene of HSP65 was amplified from DNA genome of Mycobacterium tuberculosis H37Rv strain by polymerase chain reaction (PCR). Inserted the PCR product into pMD18-T vector and sequenced. Ligated the right sequenced HSP65 gene into pPRO EX HTa prokaryotic expression vector and identified by restrictive enzyme digestion, the recombinant plasmid was named pPRO-hsp65. The recombinant plasmid was transformed into E.coli DH5αstrain and induced with IPTG.The analysis of SDS-PAGE showed that there was a specific protein expression at 65Ku molecular marker, and the protein was further identified by Western-blot using anti- HSP65 mAb and serum of TB patient. Purified the fusion protein by Ni-NTA purification system.2. Subcloned the gene of HSP65 into eukaryotic expression vector pcDNA3.1 (-), the recombinant plasmid was named pcDNA-hsp65 and identified by restrictive enzyme digestion.Transfected the recombinant plasmid into P815 cells stably by Suohua-sofast transfection reagent. The specific mRNA of the gene was detected by RT-PCR, and the expression of HSP65 protein in the transfected cells was detected by indirect immunofluorescence technique (IIFT). The result showed that there were specific strap in the agarose gel electrophoresis (AGE) and green immunofluorescence showed in the transfected P815 cells. Eight lines of transfected P815 cells stably expressing the protein of HSP65 was selected by G418.3. Immunized BALB / c mice with prokaryotic expression protein and eukaryotic recombinant plasmid of HSP65 gene, and tested the two vaccines immunological parameters. Results showed that, both the subunit and DNA vaccine of HSP65 protein could elicite great humoral and cellular immune responses, and had their own characteristics. The two vaccines could significantly reduce the bacteria loads in spleen and lungs of infected mice, this meant that the vaccines could prevent the mice against MTB infection, but the ability of protection was not better than that of BCG.All these conclusions brought some new clues for researching prevention and treatment of tuberculosis.