The Study On The Characterstic And Preliminary Application Of ClpP1Recombinant Protein Of Mycobacterium Tuberculosis

Objective：To construct a prokaryotic expression vector for ATP-dependentcaseinolytic protease proteolytic subunit (ClpP1) gene of MycobacteriumTuberculosis(Mtb), to express ClpP1recombinant protein in E.coli andpurify the recombinant protein. To investigate biological characteristics ofthe ClpP1protein by stress assays and immunize rabbits for preparing ClpP1polyclonal antibody, and then analyze polyclonal antibody antigen reactivityby indirect ELISA and evaluate its preliminary application.Methods：1.ClpP1gene from Mtb(H37Rv) genome was amplified by PCR,cloned into pET32a(+) vector, and proved by restriction digestion and DNAsequencing.2.The successfully constructed ClpP1recombinant plasmid wastransformed into E.coli BL21(DE3). The recombinant protein was inducedby IPTG, identified by Western blot and purified by affinity chromatography．3.The effects of ClpP1recombinant protein were researched by stressassays which included growth test, temperature test, hydrogen peroxide testand biological membrane test and indirect ELISA with the purified ClpP1recombinant protein as antigen.4.The purified protein was used to immune New Zealand rabbits forpreparing ClpP1polyclonal antibody and titer of the antibody wasdetermined, and then specificity of the ClpP1polyclonal antibody wasfurther detected with ClpP1protein of BCG by Western-blot test.5.The ClpP1antigen levels in serum of tuberculosis, lung cancer,pneumonia, asthma and chronic obstructive pulmonary disease patientswere determined by indirect ELISA with prepared ClpP1polyclonalantibody, and its sensitivity and specificity were calculated.Results:1.The size of restriction fragment of recombinant plasmidpET32a(+)-ClpP1was in agreement with theory and sequencing analysiswas consistent with ClpP1gene in Gen bank.2.About35KDa Specific band was found by SDS-PAGE andWestern-blot. The expressed recombinant protein was subsequentlypurified by affinity chromatography and contained about30%of total cellprotein, concentration and purity of the purified ClpP1were1.1mg/ml and93%respectively by Bandscan analysis software. 3.The stress assays researching showed that expressed recombinantprotein ClpP1could accelerate the rate of growth and improve tolerance oftemperature and hydrogen peroxide of the host strain, but could notpromote the formation of biological membrane of the host strain. Then theClpP1antibodies in serum of tuberculosis patients and healthy individualswere determined by indirect ELISA with the purified ClpP1recombinantprotein as antigen and the results found that there were significantdifferences between the two groups.4.The purified protein was used to immunize New Zealand rabbits for4times for preparing ClpP1polyclonal antibody and titer of the preparedantibody was detected as1:640000.Western-blot test showed that thepolyclonal antibody was specifically combined with ClpP1protein of BCG.5.The ClpP1antigen levels in serum of tuberculosis, lung cancer,pneumonia, asthma and chronic obstructive pulmonary disease patientswere determined by indirect ELISA with prepared ClpP1polyclonalantibody, the statistic results showed that there were significant differencesbetween tuberculosis and other diseases groups, and sensitivity andspecificity of the assay were69.9%and89.3%respectively.Conclusion:1.The prokaryotic expression vector for ClpP1gene of Mtb wasconstructed successfully and purified immunogenic ClpP1recombinantprotein was prepared. 2.The recombinant protein ClpP1has a good antigenicity and canpromote growth and improve tolerance of temperature and H2O2of the hoststrain, but does not have a promoting effect on the biological membraneformation of the host strain.3.High titer and better specificity and antigen reactivity polyclonalantibody is successfully prepared, which has a good prospect in applicationto rapid serological diagnosis of tuberculosis.