Cloning And Expressing Of Immunodominant Antigen Genes Of Mycobacterium Bovis And Preliminary Study On Nanovaccine

Mycobacterium Bovis is the causive agent of bovine tuberculosis in a range of animal species and human beings. The prevalence and circulation of this disease not only affect the development of stock raising severely, but also menace the health of human beings. Nanovaccine of bovine tuberculosis has broad application prospects, since that nanovaccine had lots of advantages, includes sustained-release, stability, targeting, and security. Thus, in this study, we prepared the new nanovaccine against bovine tuberculosis and study its immunogenicity.Prokaryotic expression plasmids of four immunodominant antigen:CFP10, ESAT6, Ag85A and Ag85B which genes were amplified from C68001chromosomal DNA were constructed by molecular cloning techniques. The recombinant protein Ag85B was induced expression by IPTG, purified by nickel column, and analysed by Western blotting. The nanovaccine was prepared by using the gelatin to wrap Ag85B protein, the drug loading and encapsulation efficiency was detected and nano-particles were characterized by TEM. Mice as experimental model, the immunogenicity of nanovaccines were studied form humoral immunity, cellular immunity and mucosal immunity.Results:(1) Constructed the four recombinant expression plasmid pET-28a-cfp10, pET-28a-esat6, pET-28a-ag85a and pET28a-ag85b, and obtaining their recombinant gene engineering strains.(2) Expressed and purifed of Ag85B protein.(3) Prepared stable nanovaccine, the average particles size of nanoparticle was uniformly about100nm. The nanovaccine drug loading and encapsulation efficiency was71.83%,41.03%respectively. Its features reached the technological requirements of the nanovaccine particles.(4) The Ag85B nanovaccine can effectively improved the expression level of antibody, IFN-γ, IL-2and slgA which can stable expressed last a long time subsequently, and also improved the thoracic spleen Index.The study suggests that the nanovaccine can induce the earlier, higher and stable expression levels of antibody and effectively induce humoral immune, cellular immunity and local mucosal immunity. This nanovaccine has good immunogenicity in effectively prevent bovine tuberculosis. This study provides a theoretical basis for preparing novel vaccine.