Preparation The Immunity Advantage Antigen PPE68 In The Mycobacterium Tuberculosis And Evaluation Of Their Immunodiagnostic Value

Mycobacterium tuberculosis is the causative agent of tuberculosis which could spread among human, fowl, badger and wildlife. Since 20th century, the tuberculosis has become more and more serious and have been the biggest killer worldwide in all of infection diseases because of population increase,human immunodeficiency virus spread and immunosuppressant usage. At present, the standard of tuberculosis diagnoses is clinical check combining mycobacterium tuberculosis cultivation and X-ray check. All the above methods depend on the clinical symptom of the patients so that the forepart subclinical infects are hardly found. Therefore, the primary measure of global tuberculosis control is quick and exact diagnoses.Looking for the specific antigen of mycobacterium tuberculosis is significant for tuberculosis diagnose.RD1 which correlates with virulence of mycobacterium tuberculosis ,is the first section lost in the attenuation process of Bacilli Calmette-Guerin(BCG).Now, the research of the mycobacterium tuberculosis diagnose reagent are mainly focusing on RDl section because that the protein which encoded by the gene of RDl could be highly recognized by the immunity system.RD1 totally encodes 9 proteins, moreover, PPE68 protein is encoded by Rv3873 in this section. It is researched that PPE68 could be recognized by lymphocytes and explode intense cell immunity to stimulate the cell multiplication of rat spleen lymphocyte with inducing amount of IIFN-y.So ,we considered PPE68 is the third important immunodominant antigen besides ESAT-6 and CFP-10.In the study,Rv3873 was amplified from H37Rv of mycobacterium tuberculosis by PCR and directed cloned into expression plasmid pGEX-4T-l.Construction the recombinants Rv3873 and expressed in E. coli JM109 induced by isopropyl—beta D—thiogalactopyranoside(IPTG) and confirmed by Western blot,then,purified the fusion protein by GST purified kit and confirmed by SDS-PAGE and Western blot.rPPE68 was used to immunize rabbit to obtain the polyclonal antibody. We set up indirect ELISA method that use PPD,rPPE68 as coating antigen to detect specific antibodies of tuberculosis patient. In order to different the patient whether for the present sickness infection, we set up double-antibody sandwich ELISA method that use polyclonal antibody as coating antibodies to detect specific antigen of tuberculosis patient and provide new technical thought for early diagnosis, early treated in tuberculosis. On the other hand,we sub-cloned Rv3873 into the eukaryotic expression vector pcDNA3.1(+)and constructin the recombinant pcRv3873,which was identified by restriction analysis and PCR. It laids a good foundation for the research of new DNA vaccine of Mycobacterium tubereulosis.The study was divided into five parts:1. the Rv3873 gene was amplified from H37Rv of mycobacterium tuberculosis DNA genome by PCR and directed cloned into expression plasmid pGEX-4T-l, construct prokaryotic recombinant plasmid pGRv3873 successfully.2. we express the rPPE68 fusion protein of approximately 63kDa in size after induced by IPTG and dissolve the inclusion body as dissolvable protein.3. Purified rPPE68 fusion protein and immune rabbit,then obtain specific,high titer of polyclonal antibody.4. We set up optimized PPD-ELISA procedure that use PPD as coating antigen, according to experiment at diluents for serum, blocking buffer, incubation time for enzyme-substrate reaction and the method of result interpretation. Similarly, we set up an indirect EL1SA with rPPE68 as coating antigen to detect the speci -fic antibodies tuberculosis patients.The result shows that sensitivity is 57% and 30% respectively,the specificity is 73.6%,92.7% respectively.According to an alyze, rPPE68 displaied great specificity but unsatisfactory sensitivity in diagn -ose of tuberculosis compares to PPD.We also set up double-antibody sandwich ELISA method that use polyclonal antibody as coating antibodies to detect speci -fic antigen of tuberculosis.The result displaied that the anti-rPPE68 has low sensitivety(only 13%),but high specificity(93.6%), therefore,we consider the EL -ISA method that detecting specific antigen of tuberculosis by specific antibody of tuberculosi is a workable new technique for diagnosing whether the tuberculo -sis is present sickness infection.5. the target gene of pGRv3873 was sub-cloned into the eukaryotic expression vec -tor pcDNA3.1(+) and construct eukaryotic recombinant plasmid pcRv3873 succ -essfully, It laids a good foundation for the further research for the immunegenic -ity and protective immunity of Rv3873 gene.In conclusion,the study chose PPE68 that encoded by Rv3873 gene of Myco -bacterium tuberculosis in RD1 as our target protein,obtained polyclonal antibody ,set up ELISA method to detect the serum of tuberculosis patient,health people and respiratory disease without tuberculosis.It is provide an important experiment data for diagnose of tuberculosis, the result shows that rPPE68 and anti-rPPE68 have little diagnosis value when use alone,but it could be used as one of the can -didate antigen to detected tuberculosis by combination antigen.