Cloning And Expression Of Heat Shock Protein 16.3 Gene Of Mycobcterium Bovis BCG

Mycobacterium is the causive agent of tuberculosis in a range of human beings. Since 1990s, the incidence of human tuberculosis has risen broadly, and the death toll has reached 3 millions every year and become the maximum among many infections. Moreover, it became the maximal record for recent years. Now, there are 5 millions tuberculoses people in our country which is one of the 22 countries for bearing tuberculosis. Together with the mobile population, multi-drug resistance (MDR) of Mycobacterium, combined infection of AIDS and many social and economic factors, they have made the diagnosis and cure more difficult and influenced the prevention of tuberculosis severely, so now people are more attentive to tuberculosis than before. WHO indicated that if a country is contaminated by tuberculosis, the human beings will always be menaced by it. If they do not take actions to eliminate it, the control of human tuberculosis will be unsuccessful. Nowadays, some developed countries and regions, such as USA, Australia and north Europe etc have eliminated bovine tuberculosis to some extents, but the prevalence of tuberculosis of human beings and wild- -life made these countries detect it all the time. In some developing countries, bovine tuberculosis still prevails severely. In recent years, with the development and application of molecular biology and immunology, we have more deeply known about the gene sequence, pathogenic and immunological mechanism of Mycobacterium than before, so the prevention of tuberculosis can base on it.Recent reports indicate an important role for M.tuberculosis (MTB) heat shock protein (HSP) 16.3 in the survival of MTB during prolonged periods of infection. It was shown that MTB HSP 16.3, initially described as the immunodominant 14- or 16-kDa antigen, was a major component in tuberculosis infection in humans and played an important role in enhancing protein stability and survival. Eighty-five percent of patients with active tuberculosis showed a positive reaction to this antigen, suggesting that this protein expressed in vivo had a key role in MTB infection .The 14K antigen was later renamed MTB HSP 16.3. MTB HSP 16.3 accumulates to become the dominant protein in the latent stationary phase of M.tuber- culosis infection. Over-expression of HSP 16.3 in log phase growth of M. tuberculosis slowed the growth rate and protected against stationary phase autolysis in vitro. MTB HSP 16.3 has been characterized as a membrane associated protein having sequence homology to other proteins in the small heat shock protein (sHsp) family. All sHsps share sequence similarity in a conserved 80-100 amino-acid"α-crystallin"domain region found in the C-terminus which is thought to be important for chaperone functions. MTB HSP 16.3 has been shown to contain an oligomeric, active structure which may form a trimer of trimers and possesses in vitro molecular chaperone activity.In this study, based on the analysis of national and international human tuberculosis, the sequential determination of HSP16.3 gene and its expression in E.coli BL21 (DE3) were the main contents, so we can study the characteristic of MTB HSP 16.3 and lay a solid foundation for the study of the latent stationary phase of M. tuberculosis infection. The HSP16.3 mature protein gene was amplified from BCG genome DNA by using PCR technique and cloned into pProEXHTb vector system. Recombinant clone was identified by enzyme digestion and sequential determination, it supported that the recombinant plasmid pProEXHTb- HSP16.3 was successfully constructed, and the sequential homogeneity reached 100%, and the amino acid did not mutate. The preceding analysis indicated that HSP16.3 gene was very conservative in BCG. The plasmid was transformed into E.coli BL21 and induced by IPTG when it was induced to 4 hours; its expression quantity reached the peak. The expression protein was analyzed by using SDS-PAGE and Western-blot to be proved that it had been expressed successful. So these results could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of mechanism of latent stationary.