Cloning Expression And Preliminary Application Of PE/PPE Specific Genes In Mycobacterium Tuberculosis

Mycobacterium tuberculosis is a pathogenic bacteria to tuberculosis, which is serious harmful to human health and our global. Along with the floating population increase, multiple drug-resistant strains emerge and HIV and MTB comorbid infection, MTB infection and spread showed a ascendant trend, has become a serious problem in public health.Early diagnosis and standardized treatment is the main control strategy, so the fast diagnosis method with specific and sensitive is urgent needed.With completion of the MTB gene sequencing and the PE/PPE protein family was found, presumably, they may have important immunology value, which can help research and development new specific diagnosis reagent and effective TB vaccine.Through the bioinformatics analysis with all of the MTB 167 PE/PPE, forecasting membrane protein and secrete proteins forecast, selecting lower homology bewteen BCG and PE/PPE, and analyzing the RD1～RD16 PE/PPE gene distribution and dominant antigen epitope, three PE/PPE purpose gene Rv3873_21～235,Rv3425_19～176,Rv2487c_421～694 as our study research object. The recombinant protein fragments with dominant antigen epitope may provide a new thought in tuberculosis diagnosis research.Experiments have constructed successfully three restructuring expression plasmid pET-28a(+)-Rv3425_19～176,pET-28a(+)-Rv3873_21～235,pET-28a(+)-Rv2487c_421～694. Prokaryotic expression and purifying protein fragments with dominant antigen epitope(rRv3873_21～235,rRv3425_19～176,rRv2487c_421～694) , and for antigenicity analysis with rCFP10-ESAT6.Western blot show good antigenicity of rRv3873_21～235, rCFP10-ESAT6 and rRv3425_19～176, but rRv2487c_421～694 has not been detected positive band.The ELISA results show the specificity and sensibility of rRv3425_19～176,rRv3873_21～235 and rCFP10-ESAT6 respectively is 39% and 97.5%, 28% and 94.6%, 37% and 95%. In addition, rRv3425_19～176 and rCFP10-ESAT6 conjoint analysis by ELISA showed that positive rate in tuberculosis patients serum is 57%(57/100), improving effectively the sensitivity of the antigen. That preliminary display a certain complementary antigen of antibody detection in TB, provided a reference with improving the diagnostic sensitivity in joint diagnostic antigen.ELISPOT results show that rRv3873_21～235 and rRv3425_19～176 didn't have spots reaction,but for rRv2487c_421～694 and rCFP10-ESAT6, the positive rate of 28 tuberculosis patients is 78.6% and 75%, the positive rate of 21 healthy persons is 19% and 23.8%. As for a antigen which have not seen reported, rRv2487_421～694 shows similar results with CFP10-ESAT6 in ELISPOT. that tip us rRv2487_421～694 may be a backup optional antigen which based on T cells in tuberculosis diagnostic tests.The epitope of gene by bioinformatics prediction, providing a reference for finding a better target site in antigen, that would have potential application value in tuberculosis diagnosis, and could be used as candidate antigens for united diagnosis.