Prokaryotic Expression Of Mycobacterium Tuberculosis Lhp Gene And Development Of Monoclonal Antibodies Against The Product

Tuberculosis(TB),a kind of chronic and consumptive zoonosis has become the top killer and the most frequent cause of death from a single infectious agent.In developing countries,TB is more intensive due to the immunodeficient people infected with virus.With the increase of population infected with HIV and the emergence of multidrug-resistant strains of Mycobacterium tuberculosis,the incidence of tuberculosis increased among the last years.Early diagnosis of infection is crucial to prevent the disease spreading,and improved methods are urgently required.Lhp which is absent both in vaccine substrain M.bovis bacille Calmette-Guerin(BCG) and in environmental nontuberculous mycobacteria(NTM) is the specific gene of Mycobacterium tuberculosis and other pathogenic mycobacteria.Lhp contributes to bacterial virulence,without the gene,Mycobacterium tuberculosis will be attenuated. CFP-10 protein was encoded by lhp gene.In this study,recombinant bacteria expressing CFP-10 of Mycobacterium tuberculosis were constructed respectively;the expressed CFP-10 protein bearing immunobiological characteristic was acquired and hybridoma cell lines secreting McAbs against CFP-10 were generated.These results would be useful for studying the pathogenesis and the immune mechanisms of Mycobacterium tuberculosis,and provided new reagent for diagnosis and quarantine.1.Prokaryotic expression of Mycobacterium tuberculosis lhp gene in recombinant E.coliThe immunodominant antigen of CFP-10 protein plays an important role in pathogenesis and immune mechanism.By PCR and GeneSOEing methods,the Mycobacterium tuberculosis lhp gene and lhp-linker-esat-6 fragment were amplified and were inserted in expressing vector through sequencing confirmation,then the recombinant bacteria BL21-pGEX-6p-1-lhp and BL21(DE3)-pET-30a (+)-lhp-linker-esat-6 were developed.With IPTG induction,the fusion proteins were successfully expressed,and the size of them were consistent with the theoretic prediction.The products of fusion proteins afforded further studies on immune functuions of CFP-10 protein.2.Production and characterization of monoclonal antibodies against CFP-10 proteinTo prepare monoclonal antibodies against CFP-10 protein,purified protein His-CFP-10,which is the product of recombinant bacteria BL21(DE3)-pET-3Oa(+)-lhp, was used as immunogen to vaccinate 8-week-old BALB/c mice subcutaneously.The immunization dose was 100μg of His-CFP-10 protein emulsified in complete Freund's adjuvant for the first immunization and in incomplete Freund's adjuvant for the next two injections with 2-week intervals.Then an intravenous dose of purified protein was administrated.After 3 days,splenocytes from immunized mice were fused with Sp2/0-Ag-14 myeloma cells.Purified GST-CFP-10 protein was used as detection antigen,and the supernatant of hybridoma clones were screened by indirect ELISA. Seven hybridoma cell lines secreting McAbs against CFP-10,named 2E7,6E8,7B11,7C2,7C3,7D4,8F6 were obtained.The immunogiobulin subclass of 6E8 and 7B11 was IgG1,and others were IgG2b.The ELISA titers of these McAbs ascites were 1×10~6,1×10~6,1×10~6,1×10~5,1×10~5,1×10~4,1×10~4 respectively.In Dot-ELISA, seven McAbs could only react with BL21(DE3)-pET-3Oa(+)-lhp,BL21-pGEX-6p-1-lhp and BL21(DE3)-pET-3Oa(+)-lhp-linker-esat-6 which expressed His-CFP-10,GST-CFP-10 and His-CFP-10-linker-ESAT-6 respectively.Western-blot analysis confirmed that seven McAbs could only react with His-CFP-10 proteins.All these results suggested that the developed McAbs 2E7,6E8,7B11,7C2,7C3,7D4,8F6 against CFP-10 have been successfully developed,which may have important application value in further studies on biological character of CFP-10 protein and diagnosis of Mycobacterium tuberculosis infection.