Establishment Of The Fast Detection Method And Expression Of FadB4/cysD Of Mycobacterium Tuberculosis

Tuberculosis is getting more and more critical since the emergence and spread of AIDS all over the world and multidrug resistant strains of M.tuberculosis in the past years. Conventional techniques, such as clinical features, histopathology, demonstration of acid fast bacilli and isolation of Mycobacterium tuberculosis from the clinical specimens, are limited for diagnosis of tuberculosis in clinically because of their speed, sensitivity and specificity. It is important to develop a new diagnosis for Tuberculosis. Nested Primers based on special insertion sequence IS6110 of M.tuberculosis were designed, which were used to amplify M.tuberculosis genome DNA. This study developed the nested-PCR diagnosis method for detecting M.tuberculosis, and test the Staphycococcus aureus, streptococcus, E.coli, Salmonella, Corynebacterium, Pseudomonas aeruginosa, Listeria monocytogenes, Brucella, et al. The sensitivity of the nested-PCR diagnosis method is detected by the ladder concentration of simulation positive sputum samples. This study developed the nested-PCR detection method for detecting M.tuberculosis, 244bp fragment was obtained as we amplify the M.tuberculosis by the nested-PCR diagnosis method,nor were Staphycococcus aureus, streptococcus, et al. The sensitivity of the nested-PCR diagnosis method is 4 copies/reaction. 41 clinical samples of different places were detected, and the positive rate was 60.98%。fatty acid metabolism and Sulfur metabolism has been implicated in the virulence, antibiotic resistance and anti-oxidant defence of Mycobacterium tuberculosis. The genome of Mycobacterium tuberculosis encodes many proteins involved in fatty acid metabolism. The Mycobacterium tuberculosis fadB4 gene, which shares strong similarity with oxidoreductases and fatty acid synthases, and its gene product displays strongly similar to NADPH quinone oxidoreductases, which serve a role as phase II detoxifying enzymes required for defence against the intake of xenobiotics. Expression of the M. tuberculosis cysD gene, predicted to encode the adenylyl-transferase subunit of ATP sulfurylase, is upregulated by the bacilli inside its preferred host, the macrophage. This study demonstrates that cysD orthologues exist in M. tuberculosis and constitute an operon whose expression is induced by sulfur limitation and repressed by the presence of cysteine, a major end-product of sulfur assimilation. The fadB4 and cysD gene was amplified by PCR with specific primers from genomic DNA of M. tuberculosis H37Rv strain. After sequenced , BL21(DE3) strain of E.coli was transformed with the recombinant vector and induced to express recombinant proteins. The relative molecular size of the proteins were analyzed by SDS-PAGE, and tested by western-blot. The fadB4 and cysD gene was cloned into PET-32a expression vector and we obtained the fusion protein fadB4 and fusion protein cysD, which molecular weight are 53kD and 55kD and possessing immunogenicity.This study developed the nested-PCR detection method for detecting M.tuberculosis, which was verified with the specificity and sensitivity. The fusion fadB4 and cysD gene and prokaryotic expression plasmid were constructed. SDS-PAGE,Westernblot confirmed the expression of the fusion proteins of fadB4 and cysD, which lays a basis for further study of natural activity and functions and its pathogenesis of mycobacterium tuberculosis.