Cloning And Characterization Of The Gene Encoding Cold-shock Like Protein A Of Mycobacterium Tuberculosis

Cold shock proteins are stress proteins found in a wide range of organisms. As molecular chaperones, they are rich in aromatic amino acids, and they bind nucleic acids, regulate transcription and translation of protein and attenuate the harmful effect of low temperature. Major cold shock protein CspA in Escherichia coli was first reported and studied thoroughly, its homologous proteins exist in most mesophile, psychrophile and thermophile bacteria. Eukaryotic cold shock domain are also homologous with CspA in Escherichia coli. Lately, increasing evidences suggest that cold shock proteins, esp. CspA have crucial functions in pathogen pathogenicity, prevalence and response for the humoral immunity system. However, the molecular mechanism of CspA in immunity and drug resistance are still unknown. The reports civil and abroad about this protein in those aspects just focused on Staphylococcus aureus. Studies about immunoreaction of CspA in Mycobacterium tuberculosis were just done in vitro. Recently, it is reported that CspA in Escherichia coli are in involved in the formation of biofilm, which indicates this protein might has close relationship with drug resistance and persistant infection of pathogens.TB has reemerged as a serious public health threat worldwide because of the increasing of multiple drug resistant TB and synergism between Human Immunodeficiency Virus (HIV) and Mycobacterium tuberculosis infection, which underscores the urgent need for the discovery of novel antimycobacterial targets and development of new drugs. The bacterium relies on a plethora of molecular chaperones to survive, including cold shock proteins. A stress protein cold-shock like protein A (CspA) in Mycobacterium tuberculosis is a culture filtrate protein (CFP), and putative protective T cell antigen with immunodiagnosis value and also subunit vaccine component against TB. In this study, cspA in Mycobacerium tuberculosis was amplified by PCR using two oligonucleotide primers, The amplified DNA was about 200 bp and was sequencing by Dingguo biotech. The amplified DNA was inserted into the vector pET 32a(+) producing a recombinant plasmid (pCspA-pET32) used to transformed into E. coli BL21(DE3) for expression of CspA. The host was induced by IPTG, and the target recombinant peptide was purified in two different ways, one is on Ni sepharose, the other is using a special approach "osmotic shock" combined with ammonium sulphate precipitation. The crude protein solution was got by osmotic shock, then precipitated at different percentages of ammonium sulphate saturation, 20％-60％. The purified protein was analysized by SDS-PAGE and ultraviolet absorption. It is found that Osmotic shock method combined with 30％of ammonium sulphate saturation, recombinant CspA concentration is the highest and the protein purity is close to column chromatography. This technique for purification of recombinant CspA is especially fit for industrial and large scale production.The proteolytic susceptibility of CspA in vitro suggested that the stabilization of CspA in vivo is mediated by binding to their substrate mRNA. Moreover, E. coli BL 21(pCspA-pET32) was more sensitive to some antibiotics, e.g kanamycin and forsee the recombinant CspA could be considered as a candidate antimycobacterial Synergist in vivo.Signal P software was used to analysize CspA of Mycobacterium tuberculosis, but none of the detectable signal sequences in the gene sequence was found, which may indicate in addition to a signal peptide, other factors has a major influence on the localization of mycobacterial proteins. Circular dichroism was used to determined the secondary structure feature of these proteins. The ellipticity in the far-UV range of CspA is lower than control (the vector protein), because the CSPs lack a-helices. The circular dichroism maximum of CspA at 197 nm originates from the antiparallelβ-sheet structure, while maximum of control at 195 nm originates from the a-helix structure. There were only a little difference between the control and recombinant protein, indicating that the inserted protein did not change the secondary structure of the vector sharply. The modeling of CspA of Mycobacterium tuberculosis was based on the tertiary structure of the CspA of Escherichia coli.The role of secretory proteins of Mycobacterium tuberculosis in pathogenesis and stimulation of specific host responses is well documented. As one of secretory proteins, CspA were analyzed computationally for the presence of HLA classⅠbinding nonameric peptides. All possible overlapping nonameric peptide sequences were analyzed for their ability to bind to alleles belonging to A, B and C loci of HLA classⅠby BIMAS. Comparative study with human proteome was used. The implication of the finding would be helpful for vaccine development.