| BackgroundTuberculosis is an ancient disease,which is the number one killer of infectious diseases,and a serious threat to human health.The diagnosis and treatment of tuberculosis remain a worldwide problem.Early rapid diagnosis of Mycobacterium tuberculosis infection is essential for the control and prevention of tuberculosis.The current clinical diagnostic methods,can not diagnose tuberculosis early and quickly.Imaging and bacteriological examination are used in clinical commonly,have low sensitivity and time-consuming,tuberculin skin test has a extremely high false positive rate.The gamma interferon release experiment(IGRA),which is currently based on cellular immunity,has been developed rapidly in recent years,but it does not distinguish between active and latent tuberculosis.It has not been found that a kind of Mycobacterium tuberculosis antigen can detect all the antibodies in all tuberculosis patients,so searching new antigen markers has become a hot issue at home and abroad.ObjectiveThe aim were to clone and express the recombinant protein Rv2991,to exploreits properties with Mycobacterium tuberculosis specific antigen,and evaluate the value of single antigen Rv2991 and composite ESAT-6/CFP-10/Rv3615 c in humoral and cellular immune diagnosis,especially for the diagnosis of smear negative pulmonary tuberculosis.MaterialsThe Rv2991 gene sequence was searched by TubercuList.The primers were designed by Primer 5.0 software.The recombinant plasmid pET28a-Rv2991 was obtained by using pET28 a and Rv2991.The recombinant plasmid pET28a-Rv2991 was transformed into BL21(DE3)PlysS competent cells for expression and purification,The recombinant protein Rv2991 was obtained,then the endotoxin was removed by phase separation methods Triton X-114,the endotoxin was measured by the methods of chromogenic substrate limulus reagent,and the target protein was measured by BCA method.The sensitivity and specificity of Rv2991 in humoral immune diagnosis were evaluated by ELISA.The mononuclear lymphocytes(PBMCs)were stimulated with Rv2991 and ESAT-6/CFP-10/Rv3615 c,SFCs were enumerated by ELISPOT,to evaluate the value in cellular immunity.Results1.The IgG levels were measured by using the recombinant antigen Rv2991,ESAT-6,CFP-10 and the composite antigen ESAT-6/CFP-10/Rv3615 c as stimulating antigens to detect IgG levels in the tuberculosis group and the healthy group.IgG levels of the four antigens in the tuberculosis group were higher than healthy group,the difference was significant statistically(P <0.05).The sensitivity of IgG antibody induced by Rv2991(24.0%),was lower than ESAT-6(32%),and equal with CFP-10(26.0%);The specificity of IgG antibody induced by Rv2991(91.4%),is higher than ESAT-6(82.9%)and CFP-10(85.7%);The sensitivity of IgG antibody induced by the composite antigen ESAT-6 / CFP-10 / Rv3615 c was higher than single antigen.2.The sensitivity of Rv2991,ESAT-6,CFP-10 and ESAT-6 combined with CFP-10 was 63.2%,73.7%,68.4% and 81.6%,respectively.The specificitywas 93.3%,90.0%,90.0% and 86.7%,respectively.The positive rates of ESA T-6 / CFP-10/Rv3615 c,ESAT-6 and CFP-10 were detected in the patients with pulmonary tuberculosis,pulmonary disease and healthy group,for any antigen s,the positive rate of TB antigen was no significant difference between pulmo nary disease with healthy group,while the positive rate of the tuberculosis gr oup was higher than the former two groups.The sensitivity of the composite antigen ESAT-6 / CFP-10 / Rv3615 c,ESAT-6 / CFP-10,ESAT-6 and CFP-10 was 87.7%(71/81),82.7%(67/81),76.5 %(62/81),74.1%(60/81),respectively.The sensitivity of ESAT-6/CFP-10/Rv3615 c was higher than ESAT-6 / CFP-10,ESAT-6 and CFP-10,and there was no significant difference between the two groups(P <0.05).The total specificity was 81.8%(54/66)?84.8%(56/66)?84.8%(56/66)and 87.9%(58/66),respectively.There was no significant differe nce between the two groups(P> 0.05).The positive rate of ESAT-6 / CFP-10/ Rv3615 c,EAST6 peptide and CFP10 peptide were 94.0%(47/50)?90.0%(45/50)?78.0%(39/50)for the diagnosis of smear positive pulmonary tubercul osis,respectively,and were 65%(13/20),45.0%(9/20)and 60.0%(12/20)in t he diagnosis of smear negative pulmonary tuberculosis,respectively.ConclusionIn this study,the recombinant protein Rv2991 was successfully cloned and the endotoxin produced by prokaryotic expression was removed and the application requirements of the compound immunoassay test were observed.The results showed that Rv2991 was expected to be a candidate antigen for tuberculosis diagnosis.The composite antigen ESAT-6 /CFP-10/Rv3615 c had a higher level of humoral immunity and cellular immune response induced by recombinant protein Rv2991 and ESAT-6/CFP-10/Rv3615 c,High sensitivity and specificity,especially for the diagnosis of bacterial vaginal tuberculosis has important diagnostic value. |
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