Cloning, Expression And Preliminary Application Of Specific Antigen Rv0315and Rv2971from Mycobacterium Tuberculosis

BackgroundAs the sharp deterioration of TB situation worldwide, more and more peoplewere caught by TB both in developing and developed country by the contribution ofAIDS spreading, sorts of DR-TB increase, immigration as well as neglection of TBcontrol in many countries and regions etc. Fast&accurate diagnose onmycobacterium TB infections and particularly on negative TB and latent TBinfections could make great contribution to TB control. On this account, the TBcontrol would highly be influenced by the diagnostic level. Searching fast&accuratediagnose methods has been a hot issue at home and abroad owing to presently clinicallimitations like low smear detection rate, long training cycle and so forth.ObjectivesThe aims were to develop new markers for TB diagnosis, to get specific antigensRv0315and Rv2971by gene clone and expression, and to evaluate the diagnosisvalue of single antigen and complex antigen both in humoral immunity and cellular immunity.MethodsThe complete genome sequence of mycobacterium tuberculosis was analyzedand the primers of Rv0315and Rv2971were designed. Recombinant plasmidpET21a-Rv0315and pET32a-Rv2971were developed through molecular cloningmethods. Then the recombinant proteins were reached after conversion following byexpression and purification. The endotoxin would be removed by phase separationmethods Triton X-114. The concentration cells and antigens were established byELISPOT. After evaluating the value of Rv0315, Rv2971, EAST-6、CFP-10、ESAT-6/CFP-10/Rv0315with ELISA, their sensibility and specificity amid humoralimmunity were got. Method of ELISPOT was consulted to assess the value ofRv2971and complex antigen ESAT-6/CFP-10/Rv0315amid cellular immunity.Results1. The recombinant plasmids of pET21a-Rv0315and pET32a-Rv2971weresuccessfully constructed, and even recombinant proteins were gained aftertransformation following by expression and purification. Besides, the endotoxincontained was lower than5EU/mg.2. After detection by ELISA, the sensibility of Rv0315, complex antigenESAT-6/CFP-10/Rv0315, ESAT-6, CFP-10, Rv2971were20.8%(10/48),37.5%(18/48),31.3%(15/48),25%(12/48),4.2%(2/48), respectively in tuberculosis group.Specificity were93.8%(30/32),87.5%(28/32),87.5%(28/32),90.6(29/32),90.6(29/32), respectively in health control group.3. Through ESLIPOT, the positive rate of complex antigen Rv0315, Rv2971,peptide of EAST-6and CFP-10were58.3%(14/24),16.7%(4/24),75.0%(18/24),66.7%(17/24). Specificity were92.9%(13/14),85.7%(12/14),92.9%(13/14),92.9%(13/14).4. The comparison of positive rate between Rv0315and ESAT-6were correctedχ2=0.900, P=0.344; and CFP-10was corrected χ2=0.125, P=0.727. The comparison ofpositive rate between Rv2971and ESAT-6were corrected χ2=10.564, P=0.001; and CFP-10was corrected χ2=6.667, P=0.007. Through ESLIPOT, the positive rate ofcomplex antigen ESAT-6/CFP-10/Rv0315, peptide of EAST-6and CFP-10were85.7%(60/70),77.1%(54/70),72.9%(51/70). Specificity were82.4%(28/34),85.3%(29/34),88.2%(30/34). The comparison of positive rate betweenESAT-6/CFP-10/Rv0315and ESAT-6were corrected χ2=4.167, P=0.031; and CFP-10were corrected χ2=7.111, P=0.004. The positive rate of ESAT-6/CFP-10/Rv0315,ESAT-6, CFP-10were94.0%(47/50),90.0%(45/50),78.0%(39/50), respectively indiagnosed smear positive pulmonary tuberculosis group. In diagnosed smear negativepulmonary tuberculosis group were65%(13/20),45.0%(9/20),60.0%(12/20),respectively.Conclusions1. The endotoxin generated from pronucleus expression would be separated bythe method of Triton X-114, what’s more, the endotoxin content met the immune testapplication requirement.2. MTB specific antigen Rv0315is expected to be a novel candidate antigen forthe diagnosis in TB. Rv2971, not having potential immunogenicity, is not suit toapply as a biomarker for diagnosis of tuberculosis.3. MTB specific antigen Rv2971, not having potential immunogenicity, is notsuit to apply as a biomarker for diagnosis of tuberculosis.4. The complex antigen ESAT-6/CFP-10/Rv0315potentially would be theassistant diagnoses of TB, and is deserved further research.