| Tuberculosis is a leading killer of the infectious diseases around the worldwide. Studingof pathogenesis of tuberculosis is particularly important for prevention and controllingof tuberculosis. Rv0266c from Mycobacterium tuberculosis genome was predicted toencode a5-oxo-prolinase, also named OplA, which can catalyze5-oxo-proline togenerate L-glutamate, and hydrolysis of one molecule of ATP to generate ADP and Pi.This reaction is a critical component of in the GSH cycle exsisting ubiquitous ineukaryotes. Studies about5-oxo-prolinase of about prokaryotic organism are rare. Thisstudy is aim to explore the OplA function of mycobacterium tuberculosis ideas for thestudy of tuberculosis pathogenesis. Methods：Gene cloning, expressing technology,refolding of inclusion body, affinity chromatography and SDS-PAGE were used toobtain soluble and purified expression product. The open reading frame of oplA wasknock out by technology of double-crossover homologous recombination. The growthcurves of WT,△OplA, and OplAcom, were established to study the relationship betweenOplA and growth of Mycobacterium tuberculosis. THP-1cells infection models wereset up to determinate whether OplA is relevant with bacteria virulence. Results：Solublepurified OplA was failed to be obtained through trying to optimize various parametersof the expression and purification system. The open reading frame of oplA was knockout from Mycobacterium tuberculosis genome successfully. According to the growthcurves,△OplA strain grew better than WT starin in the later culture period. Accordingto the THP-1infection models, there was not extremely obvious diference aboutvirulence among strains of WT,△OplA, and OplAcom. Conclusion：Complex foldingstructure and high molecular weight of OplA may be the key factors influencing theseparation and purification of the expression product. Under aerobic condition, thegrowth of Mycobacterium tuberculosis was not dependent on OplA. There were not enough evidence to show that OplA play an important role on bacteria virulence ofMycobacterium tuberculosis. It remains further study to verify those results. Backgrounds: China is a tuberculosis high burden country, female genital tuberculosisreveals a public health problem which cannot be ignored. Female genital tuberculosisis one of the main reasons that cause infertility. Many limitations exist in those currentdiagnostic methods for female genital tuberculosis. Comparing with other FGTBdiagnostic methods, there are many advantages on serological diagnosis, such as simpleoperation, low risk, easy to spread. Previous studies have reported that HBHA is closelyrelated with the occurrence of extra pulmonary tuberculosis. Object: The object of thisstudy was to investigate the application of heparin-binding haemgglutinin adhesion(HBHA) in prompt diagnosis of female genital tuberculosis. Methods: Therecombinant HBHA produced in E. coli BL21(DE3) was purified by affinitychromatography and characterized by Western blotting. Sera samples were collectedand screened to be recruited as objects to be measured. Then enzyme-linkedimmunosorbent assay (ELISA) was performed to detect rHBHA-specified antibodiesIgG and IgM. Then all data were collected and analyzed through two statistical analysissoftware SPSS21.0and GraphPad Prism5.0. ROC curves were set up by GraphPadPrism5.0. Results: High purity recombinant protein HBHA was obtained throughaffinity chromatography, and its strong antigenicity was proved by immunization ofmice as well as the Western blotting assay. A total of178serum samples from4groupsas following: FGTB (female genital tuberculosis) group with36confirmed FGTB cases,health control group consisting of47PPD negative subjects and47PPD positivesubjects, OGD (other types of gynecologic diseases) group with48cases confirmed asother types of gynecologic diseases except for tuberculosis. The receiver operatingcharacteristic (ROC) curve and cut-off value of IgG and IgM assays diagnosis of FGTBwere established according to concentrations of antibodies between FGTB subjects and PPD negative subjects. The sensitivity, specificity, AUC, PPV, NPV, false negative rate,and false positive rate for IgG assay were72.22%(95%CI,54.81%to85.80%) and93.62%(95%CI,82.46%to98.66%),0.86(95%CI,0.78to0.94),89.66%(95%CI,73.62%to96.42%),81.48%(95%CI,69.16%to89.62%),27.78%,6.38%, respectively.The sensitivity, specificity, AUC, PPV, NPV, false negative rate, and false positive ratefor IgM assay were47.22%(95%CI,30.41%to64.51%),93.62%(95%CI,82.46%to98.66%),0.74(95%CI,0.63to0.85),78.79%(95%CI,62.25%to89.32%),80.39%(95%CI,67.54%to88.98%),8.51%,52.78%, respectively. Conclusion: In our study,through comparing IgG and IgM assay, we suggested an ELISA assay based on anti-HBHA IgG detection with high sensitivity and specificity indicating a potential methodapplied in diagnosing FGTB disease. |
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