Cloning And Prokaryotic Expression Of HSP65Gene And HSP70Gene Of Mycobacteria Intracellular

Mycobacreria intracellular is a special kind of bacteria of nontuberculous mycobacteria. It mainly causes chronic lung infection of patients, lymphadenitis of children. And mycobacteria intracellular is usually easy to lead to disseminated infection with ADIS, so immunosuppressed patients could be infected. The researchers find that T cells mainly attack HSP when the immune reponse against mycobacteria. It is reported that there are about more than20kinds of infectious diseases which could lead to immune responses against HSP, and the most is the HSP60family. When immunologic cross-reactivities of mycobacteria occur, the protective effect of HSP65is better than the other heat shock protein. Mycobacteria HSP70also have good immunogenicity, and they can induce strong cellular reponses. At present, the studies about mycobacteria intracellular are still in the early stages, but little in the heat shock protein of mycobacteria intracellular. So, this study mainly researches the cloning and prokaryotic expression of heat shock protein65gene and70gene of mycobacteria intracellular ATCC13950which saved in the lab.In this study, the outer primes and the inner primes are respectively designed to amplify HSP65gene and HSP70gene by nested PCR. First, the outer primes of HSP65and HSP70are respectively used to amplify the outer gene fragments which used mycobacteria intracellular ATCC13950genomic DNA as templates. After the PCR products are purified, the inner primes are respectively used to amplify the inner gene fragments which used the respective outer gene fragments as templates. The PCR products which are purified are inserted into PMD18-T vector to construct the recombinant cloning plasmids which called PMD18-T-HSP65and PMD18-T-HSP70. And then they are transformed into E.coli cells DH5a, cultured55min at37℃, and then paint on the LB agar plates containing Amp+, culture15h at37℃. Pick white bacteria which grow on the LB agar plates containing Amp+randomly to LB liquid medium containing Amp+, cultured13h at37℃. Draw3ml bacteria to the centrifuge tube in order to extracte the plasmids. The plasmids are identified by PCR and Nde I, BamH I restriction enzyme analysis. The positive recombinant plasmids are sent to Sangon Biotech (Shanghai) for sequencing. The results show that the homology of ATCC13950HSP65and ATCC13950HSP70comparaed to mycobacteria intracellular in genebank are99.88%and99.95%, respectively.The PMD18-T-HSP65and PMD18-T-HSP70are digested by Nde Ⅰ and BamH Ⅰ, and cloned to PET15b vector which are digested by the same restriction endonucleases. And then transformed into E.coli cells DH5a, then cultured on the LB agar plates containing Amp+and LB liquid medium containing Amp+, and then extracte the plasmids. The recombinant plasmids are identified by PCR and double digestion. The positive recombinant plasmids PET15b-HSP65and PET15b-HSP70are transformed into E.coli cells BL21(DE3)plyss, cultured on the LB agar plates containing Amp+and LB liquid medium containing Amp+, then draw100μL bacteria to10ml LB liquid medium containing Amp+, cultured1.5h at37℃. The recombinant bacteria are induced4.5h by IPTG which final concentrztions are0.8mmol/L at37℃. The expressed products are analyzed by SDS-PAGE. The results show that the HSP65protein and HSP70protein are both well expressed in E.coli. The HSP65protein and HSP70protein are65kD and70kD, respectively. Western blot results show that the target proteins have good reactogenicities, they can react with the positive serum of mycobacteria intracellular. Which lay the foundation for the application of HSP65protein, HSP70protein and mycobacteria intracellular in the clinical diagnosis, as well as providing a theoretical basis for the deeply study of mycobacteria intracellular and HSP65, HSP70.