| Objetive:(1). By constructing overexpression strains of mazEF3,mazEF6 and mazEF9 in Beijing sublineage,no-Beijing sublineage and Mycobacterium tuberculosis standard strain(H37Rv),Observing its ability to survive under the condition of low oxygen and nutrient deficiency, at the same time testing the mRNA expression level of overexpression of strains under different voltage.(2). To investigate the possibility of seven cytokines about its mRNA expression level as biomarkers for diagnosis of latent TB infection.Methods:(1). Using the shuttle-plasmid pMV361, we connected the mazEF3,mazEF6, mazEF9 with the pMV361 to construct a recombination plasmid pMV361-mazEF3; pMV361-mazEF6;pMV361-mazEF9.The shuttle-plasmid pMV361-mazEF3; pMV361- mazEF6;pMV361-mazEF9 was transfered into H37 Rv,Beijing sublineage and no-Beijing sublineage by electroporation technology, and then we inoculated the bacteria and screened initially on kanamycin culture medium. We extracted positive strain’s plasmid, after PCR and double enzyme digestion technology, sent the PCR and enzyme digestion products to company for further verification.By the real-time fluorescent quantitative PCR, we tested the mRNA expression levels of mazEF3 under different voltages with electrotransformation in order to finding the optimal voltage.(2). Totally 64 healthy control individuals, 50 active tuberculosis patients and 60 latent tuberculosis infection individuals. For above the three groups, the mRNA expression levels of TNF-α, IFN-γ, IL-2, IL-10, IFI35, IP10 and Foxp3 in peripheral blood stimulated by tuberculosis-specific antigen were detected using real-time quantitative PCR(qRT-PCR).Results:(1). Mycobacterium tuberculosis standard strain(H37Rv), Beijing sublineage and no-Beijing sublineage overexpressing mazEF3,mazEF6 and mazEF9 gene was successfully constructed. The growth status of beijing sublineage of mycobacterium tuberculosis in normal and stress environment were better than no-Beijing sublineage. At the 2300 V with electrotransformation voltage, the mRNA expression level of mazEF3 is the highest.(2). The mRNA expression levels of TNF-α, IFN-γ, IL-2, IL-10, IFI35, IP10 and Foxp3 in the active TB group were 2.33±0.09, 4.94±0.45, 150±17.82, 2.02±0.55, 9.53±5.96, 5.56±0.97 and 1.24±0.17 respectively. The levels of these cytokines in latent tuberculosis infection groups were 3.2±0.61, 10.04±1.89,202±17.6, 21.89±10.25, 29.17±11.19, 41.35±11.46 and 2.14±0.67 separately. The value of healthy group was1.2±0.08, 2.92±0.03, 53±17.82, 1.48±0.17, 1.28±0.77, 1.63±0.25 and 1.03±0.67 in sequence. Between latent infection group and healthy control group, the 7 cytokines’ s mRNA expression levels were significant difference(P<0.01), and the sensitivities and specificities of the 7 cytokines were 95.1%, 82.5%; 92.5%, 88.7%;91.9%, 90.1%; 88.2%, 51.5%; 74.2%, 61.2%; 81.8%, 73.4%; 80.4%, 51.3%, respectively. Compared the latent infection group with active TB group, except IFI35 factor, the remaining 6 cytokines’ s mRNA expression levels were significantly different(P<0.05), the specificities and sensitivities of the 7 cytokines were 90.6% and 80%;88.6%, 92.7%; 91.9%, 86.2%; 57.6%, 60%; 50%, 58%; 76%, 72.2%; 66.7%, 67.1% separately. Compared active TB group with healthy controls group, the 7 cytokines’ s mRNA expression levels were significant difference(P<0.01), and the sensitivities and specificities of active TB group are 92.3%, 90%; 88.6%, 86.7%;94.4%, 86.7%; 89.7%, 65.8%; 82.4%, 63.3%; 88.2%, 70%; 92.9%, 72.1% respectively. Latent infection rate in high risk group was 48.97%, which in health group was 36%.Conclusions:(1). Overexpressing strains of mazEF3,mazEF6,mazEF9 gene in H37 Rv,Beijing sublineage and no-Beijing sublineage was successfully constructed. At the 2300 V with electrotransformation voltage, the expression level of mazEF3 is the highest.(2). TNF-α, IFN-γ and IL-2 mRNA express higher in TB patients,especially latent infection increased more significantly, Therefore they can be used as biomarkers for diagnosis Mycobacterium tuberculosis latent infection. |
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