Comparative Proteome Analysis Of Mycobacterium Tuberculosis Of Two Genotypes

Background: Bovine tuberculosis(TB) has not only caused tremendous economic losses to the milk industry, but also posed a serious threat to public health and safety. Recently the transmission of human tuberculosis and bovine tuberculosis causes great problem to the complete eradication of tuberculosis. Two strains of Mycobacterium tuberculosis XB07001 and XB07002 were separated and identified from the same cow herd in the northwest. Genotyping was performed using the spoligotyping technique,and the two strains belong to different genotypes, that is , Mycobacterium tuberculosis XB07001 belongs to Haarlem3 Family, while Mycobacterium tuberculosis XB07002 belongs to family 33 Family. The Haarlem 3 strain family has often been associated with tuberculosis (TB) outbreaks and drug resistance, besides Beijing strain family.Methods and Results: In this study, the proteome of two-dimensional electrophoresis combined with mass spectrometry was used to analysis the differently expressed protein of these two strains of Mycobacterium tuberculosis XB07001 and XB07002. Extracted the total soluble proteins from the two strains of Mycobacterium tuberculosis, separated proteins though two-dimensional electrophoresis technology, stained proteins with Coomassie brilliant blue , and then scanning to get the patterns. The differentially expressed protein spots was analyzed by the software ImageMaster 2D Platinum 6.0, and twenty-six differentially expressed protein spots were selected and identified by MALDI-TOF-TOF mass spectrometry. There are 21 protein spots were successfully identified, including 17 up-regulated protein spots in XB07001 and 4 up-regulated protein spots in XB07002. The 17 up-regulated protein spots are hypothetical protein Rv2226, benzoquinone methyltransferase, rRNA methyltransferase, I-type restriction modification system DNA methylase, cold shock box protein A, electron transfer flavoproteinαsubunit, hypothetical protein MT1685, hypothetical protein BCG₁806c, hypothetical protein MtubE₁1019, hypothetical protein Rv1636, short-chain dehydrogenase, superoxide dismutase SOD, pyruvate dehydrogenase, glycosyltransferase, hypothetical protein TBCG₀1580, transcription regulatory protein and malate dehydrogenase. The 4 up-regulated protein spots include elongation factor Tu, transmembrane transport protein MmpL13B, chaperonin GroES, and cell division protein FtsZ. Differentially expressed proteins were involved in methylation, energy metabolism, molecular chaperones, virulence protein and so on. 7 corresponding genes of the differently expressed proteins were chooseed to validate the results of the 2-DE by PCR and the PCR results were partial identical with 2-DE analysis. The FtsZ,GroES and SOD gene were amplified by PCR and cloned into expression vectors pET32a. The recombinant vectors pET32a-FtsZ,pET32a-GroES and pET32a-SOD were transformed into Escherichia coli BL21 (DE3 ), and then induced by IPTG. The expressed protein was purified and rabbits were immunized. Three kinds of rabbit polyclonal antibodies were obtained.and used to further analyze protein profile through western-blot and the results were consistent with the 2-DE analysis. These results show that proteomic analysis can be valuable for investigating differences in Mycobacterium tuberculosis and may provide a better understanding of the research of mechanism of pathogenesis and drug resistance.