| Background of study and Objectives Tuberculosis(TB) is a Chronic infectious disease by mycobacterium tuberculosis,which were incidenced in all over the body of human and animals organ. In recently years, drug resistant strain of TB appearance,merging AIDS and MTB and flowing of high-risk group make TB epidemic in global. Chemotherapy were main measure which TB was prevented and therapied, but long time of therapy, high cost, more strengthed side effect and appearance of persister lead to lower healing rate and higher recurrence rate. Therefore BCG has a lot of shortage, which was used widely at present. So it was very essential that new vaccine was exploitated.Esat-6 and Mpt64 were a secretion proteinum with more strengthed immunocompetence in culture filter liquor of earlier period. But they were lacked in BCG, so they were purpose gene in gene vaccine.At present, phoP gene was closely attended by plenty of researcher, which was relation with virulence of Mycobacterium tuberculosis[25,26]. It was corresponded with Rv0757 of Mycobacterium tuberculosis genome[53],and encoded transcription factor was named two-component system. The phoPR was Virulence controlling gene of Mycobacterium tuberculosis[31]. In this study, for seeking a MTB mutant strain of strengthed immunogenicity and hypotoxicity. Genes of phoP were comp lately deleted and Esat-6,Mpt64 gene recombination strain were constructed successfully byλ-Red homologous recombination. For exploitating new generation tuberculosis vaccine settle foundation.Matetials and methods 1,Strain and Plasmid origin.Standard strain H37Rv: conserved by ourselves laboratory. Plasmid pKD4 and pKD46 were honored by Dr. Han Yanping who is from the Chinese People's Liberation Army 309 hospital entire armed forces institute of microorganism epidemic.2,Culture of Bacterial Strains H37Rv were grown L-J medium modified medium and sauton medium.3,reparation of Linearity DNA frag.The Esat-6 and Mpt64 genes were amplified by PCR from MTB H37Rv. The Kan-p was priming gene and the phoP-K was kanamycin resistance gene with replacing phoP. The Kan-p and phoP-K genes were amplified by PCR from pKD4. The Kan-p and Esat-6,Mpt64 genes were connected by overlapping PCR, and the Kan-Esat-6 and Kan-Mpt64 genes were obtain.4,Electrotransformation.Electrotransforma- tion parameter: 2.5KV,25uF,200Ω; When H37Rv strain were cultured exponential phase of growth, freshly prepared competent cell were electroporated in the presence of 1ug of plasmid pKD46. H37Rv with pKD46 of exponential phase of growth was induced 48 hour with 10mmol/L L-arabopyranose, which was prepared freshly prepared competent cell, and was were electroporated in the presence of 500ng of vector DNA. Gene recombination strain of Esat-6 and Mpt64 were screened by common L-J flat in 26℃and shortcoming strain of phoP gene were screened by L-J flat with kanamycin in 26℃.5,Identify.Strain DNA was obtained By boiling method,production of DNA was amplified by Homo-arm-F and Homo-arm-R primer, and was identified by agarose gel electrophoresis and sequencing.Results To test under microscope result of H37Rv with culturing L-J and sauton medium Ziehl-Neelsen acid-fast stain.Kan-Esat-6,Kan-Mpt64 fusion gene and phoP-K identified by Agarose gel electrophoresis was about 852bp,1248bp,1678bp bp.It was confirmed that the plasmid pKD46 was successfully introduced into H37Rv by analyzing the resistance selected in L-J medium with 50μg/ml Amp by PCRIt was confirmed that the genes of phoP were comp lately deleted and Esat-6,Mpt64 gene recombination strain were successfully constructed by analyzing the resistance selected in L-J medium with 50μg/ml kanamycin and L-J medium by PCR, which was in accordance with the expected result by sequence analyzing. Conclusions Genes of phoP are complately deleted and Esat-6,Mpt64 gene recombination strain are constructed successfully. |
PDF Full Text Request