1.Studying On The Molecular Chaperone Activity And Associated Function Of Small Heat Shock Protein, Hsp16.3, From Mycobacterium Tuberculosis 2.Studying On Gene Knock Out In Mycobacterium Bovis BCG

Tuberculosis is one of the major infectious diseases, caused by the mycobacterium tuberculosis(MTB), menacing overly human lives. The ability of M. tb to grow and persist in the macrophage is one of the factors that lead to tuberculosis. It is confirmed that MTB, which survives in the macrophage, can expresses a kind of small heat shock proteins, Hsp16. 3. The cell wall will be significantly thickened while the Hsp16.3 is expressed in the cell. According to these, we suppose that the expression of HsplG. 3 be helpful to MTB surviving in macrophage.The monomer of Hsp16.3 can be spontaneously formed the nonamer, trimer of trimers, in the native state. Its function, in vitro, is mainly molecular chaperone-like activity. We proposed that the nonamer dissociation of Hsp16. 3 be prerequisite for its exhibiting chaperone-like activity and the essential unit for its chaperone-like activity be trimer according to the results of, by comparison with Hsp16. 3 and its mutation (using the method of site-directed mutagenesis) which tryptophan was substituted for the Glycine in the region of LPGV localized at the alpha-crystallin domain, the chaperone activity detection and ANS fluorescence and CD spectra and native-pore gradient PAGE and FPLC and intrinsic-fluorescence. Meanwhile, we also postulated that the region of LPGV Iodize at the action surfaces between trimer and trimer.When Hsp16. 3 and 38kDa protein were fused and expressed induced by IPTG gradient concentration, we found that the fusion protein exhibited soluble state at 0. 8mM of the final concentration of IPTG. However, 38kDa protein was still expressed as inclusion boby at the same final concentration of IPTG. So, we speculated that Hsp16.3 maybe possess the ability of intramolecular chaperone for other proteins.It is the fact that three methionines distributed in the Hsp16.3 will begradually oxidated, in vitro, by hydrogen peroxide(H2O2). When the hsp16. 3 gene was cloned with pMV261 plasmid vector and transfected into Mycobacterium smegmatis(M.smeg.), we found that the ability of anti-H2O2 will be decreased greatly among the strains of M. smeg. containing pMV261-hspl6. 3 while compared with the control strains of M. smeg. containing free plasmid vector. But we found that the ability of anti-H2O2 will be increased greatly among the strains of M. smeg. containing pMV261-38kDa while compared with the same control. The conclusion will be believed that Hsp16. 3 inside of cell has not ability to anti-H2O2How is the function of Hsp16. 3 performed in vivo? Up to the present, it is unclear yet. Although we can not screen out the substrate(s) of Hsp16. 3 by the co-immunoprecipitation(CIP), we find the facts that Hsp16. 3, which is purified from Escherichia coli(E. coli), is always accompied by the 30kDa protein. Meanwhile, the molecular chaperone-like activity of Hsp16. 3 has been clearly proven in many experiments. So, we are convinced of the substrate(s), localized at the stressed cell, interacted with Hsp16. 3.In addtion, we knocked out the MDP1 gene from BCG strain using the pKO plasmid. But it is not observed that the growth rate of the strian knocked out MDP1 gene increases. It is maybe concluded that the MDP1 gene is not only one factor to determine the growth rate. From this experiment, we have set up a technological platform for knocking out gene in the Mycobacteria.