The Coding Gene's Expression Level And Significance Of Protein In Different Mycobacterium Tuberculosis

Objective By using quantitative real-time polymerase chain reaction (qRT-PCR) method, The coding gene's expression levels of Ag85B,38kDa and MPT64 (fbpB, pstsl, mpt64) in Mycobacterium tuberculosis were detected in order to explore the variability and significance of these proteantigens in transcriptional level between the drug-resistant strains and the sensitive strains, between the Beijing genotype strains and non-Beijing genotype strains.Methods The specific primers of fbpB, pstsl, mpt64 and reference gene Siga were designed according to GenBank database. The fbpB, pstsl, mpt64 and Siga fragments were cloned to pMD18-T simple vector respectively, and recombinant plasmids were purified and gradient dilution. We established the qRT-PCR method for detecting the expression levels of fbpB, pstsl, mpt64 by using SYBR GreenⅠ, and then selected 46 Mycobacterium tuberculosis strains to do study. The 46 strains included the sensitive strains (24), the drug-resistant strains (22), the Beijing genotype strains (26), the non-Beijing genotype strains (20) and the Nontuberculosis mycobacteria(2). The expression levels of fbpB, pstsl, mpt64 in 46 Mycobacterium tuberculosis, standard strain H37Rv and 2 Nontuberculosis mycobacteria were detected by qRT-PCR method.Results By contrast to the sensitive strains and H37Rv, the expression level of mpt64 gene in drug-resistant strains was downregulated, and had statistical significance (t=3.093, p<0.01; t =2.545, p<0.05). Compared with the sensitive strains and H37Rv, the expression levels of fbpB and pstsl genes in drug-resistant strains hadn't statistical significance; The expression of pstsl gene in Beijing genotype strains was lower than that of non-Beijing genotype strains and H37Rv, the result had statistical significance(t=3.093, p<0.01; t=2.545,p<0.05). The expression levels of fbpB, mpt64 genes in Beijing genotype strains by contrast to non-Beijing genotype strains and H37Rv were no difference. The amplification results in live Mycobacterium tuberculosis shew the fluorescence signal of S curve, the ones in the death and Nontuberculosis mycobacteria appeared the fluorescence signal of a line.Conclusion This study found the expression level of fbpB gene between the drug- resistant strains and the sensitive strains, and between Beijing genotype strains and non-Beijing genotype strains were no difference; The expression level of mpt64 gene in the drug- resistant strains was downregulated compared to the sensitive strains and H37Rv, but the expression level of mpt64 gene in Beijing genotype strains and non-Beijing genotype strains was no difference;The expression level of pstsl gene in Beijing genotype strains was downregulated by contrast to non-Beijing genotype strains and H37Rv, but the expression level of pstsl gene in the drug- resistant strains and the sensitive strains was no difference.