Fused Prokaryotic Expression And Evaluation Of Immunoprotective Potency Of HBH A-HSP65from Mycobacterium Bovis

Bovine tuberculosis is one of chronic consumption of infectious zoonotic diseases causedby Mycobacterium bovis. It is classified as a must-be-reported disease by OIE, while listed it tothe2nd kind of quarantive animal disease in China. In our country, tuberculosis is an enormousdisease burden. As regards as the prevention of bovine tuberculosis, BCG isthe only vaccine thatcan be used, but after the incubation of BCG, the immunized effects is not obvious and we can’tdifferentiate the natural infection and artificial immunization. Moreover, it made the routinequarantine mare difficult.So the new vaccine for the diagnosis and prevention of the bovinetuberculosis has been the hot spot. HBHA and HSP65(Cpn60.2) were the mainly adhesion ofMycobacterium.1. Tandem expression of HBHA and HSP65. Currently, HSP65and HBHA gene was amplifiedfrom Mycobacterium bovis Vallee III chromosomal DNA by using PCR technique, and theirlength were1600bp and600bp. After sequencing correctly, HSP65, HBHA and HBHA-HSP65gene was subcloned into pET28a(+) procaryotic expression system, the plasmid was transformedinto E.coli BL21(DE3) and induced by IPTG. By SDS-PAGE, the expression protein was65kDa,28kDa,95kDa. The expression protein was analyzed by using Western-blot a to be proved that ithad the antigenic activity of Mycobacteriurn bovis.2. Subunit vaccine immunoprotection evaluation of tandem expression of HBHA and HSP65.The antigen had been emulsified with Freund’ s complete adjuvant, subunit vaccines weregenerated. After immunized mice, HBHA-HSP65antibody serum titer were detectided byELISA antibody levels was1:16000, which significantly higher than the above HBHA, HSP65and BCG group. Cellular immunity was analyzed by MTT, HBHA-HSP65immunized groupwas significantly higher than the other three groups (P <0.05).The results showed the bacterialoads of spleens and lungs in PBS group were5.67士0.11log10and5.52士0.09log10respectively, while those of in HBHA-HSP65group were4.53士0.05log10and0.21士0.02log10respectively, show that the fusion protein vaccine can significantly reduce Mycobacteriumtuberculosis load in the mouse spleen (P <0.05), almost completely cut off lung infection (P<0.01). The two vaccines could significantly reduce the bacteria loads in spleen and lungs ofinfected mice, this meant that the vaccines could prevent the mice against MTB infection, but theability of protection was not better than that of BCG.