The Mycobacterium Tuberculosis Tb32ag, Mpt64ag The Cloning And Preliminary Expression

Mycobacterium tuberculosis is considered to be one of the major causes of tuberculosis. Finding out specific antigens is very important for developing an accurate and rapid diagnosis method. Antigens TB4 of Mycobacterium tuberculosis,with molecular weights from 32KD to 40KD,also including 28KD and 23KD~25KD antigens, are associated with the specific diagnosis of tuberculosis,with sensitivity 0.9012 and speciality 0.9639. So,antigens TB4 have high application values in diagnosis of tuberculosis. According to traditional methods, antigens TB4 are obtained from Mycobacterium tuberculosis H37Rv strains by small-scale, which is very difficult for human beings because of the danger. Now, by DNA recombinant method,we have overcome all the difficulties.In this study,total DNA was extracted from M. tuberculosis H37Rv strains. According to the gene sequences of antigens TB4, we have devised several pairs of primers. By polymerase chain reaction(PCR),using the total DNA as a template, we have multiplied the genes of TB32Ag and MPT64Ag. The nucleic acid sequences of TB32Ag and MPT64Ag are analysised by Sanger's dideoxy mediated chain termination method. By sequencing, we get to know that TB32Ag gene consists of 586bp and encodes 189 amino acids while MPT64Ag gene consists of 644bp and encodes 214 amino acids.In order to express antigens TB4 in E.coli, TB32Ag gene and MPT64Ag gene are connected with the pronucleus expression vector pPROEX HTa and transformed into E.coli DH5a strains, and positive strains were induced by IPTG. And we applied SDS-PAGE gel to detect the expressed proteins. The result shows that, recombinant strains with TB32Ag gene have expressed four kinds of fusion proteins, which have molecular weights of 28KD,24KD,19KD and 16KD. Recombinant strains with MPT64Ag gene have expressed a new protein bond appeared with a molecular weight 33KD. In order to appreciate the protein immunity, the expressed protein was transferred into PVDF filter, then the polyclonal antibody was added. The result of immunoblot shows a specific immunocomplex bond in accordance with the expressed protein, which indicates that the expressed protein has immunity of TB.This study offers certain base for the research of antigens TB4 in tuberculosis diagnosis.