Construction Of The Fusion Gene Of Mycobacterium Tuberculosis Hsp65 And EGFP And Preparation Of Dendritic Cell Vaccine

Currently the main target of Mycobacterium tuberculosis research is to construct new effective anti-tuberculosis vaccine. There are high intensity antigen presenting moleculars, co-stimulate moleculars on the surface of dendritic cells which can stimulate the activation and proliferation of naive T cells. Hsp65 (heat shock proteins) is a prominent antigen in triggering and enhancing immune response to tuberculosis. Mycobacterium tuberculosis H37Rv Hsp65 DNA was cloned into eukaryotic expression vector pEGFP-C1 to construct recombinant plasmid pEGHsp65. Then pEGHsp65 was transfected into Hela cells and Hsp65 protein expression was detected. Recombinant plasmid was transfected sequently into dendritic cells to construct anti-tuberculosis new type vaccine.In this research, a pair of PCR primers were designed according to Mycobacterium tuberculosis Hsp65 gene sequence and the Hsp65 fragment was obtained from the complete genome DNA of Mycobacterium tuberculosis standard virulent H37Rv strain by PCR amplification. The purified PCR product and eukaryotic expression vector pEGFP-Cl was digested by restrictive enzyme Bgl II and EcoR I and then ligated . The recombinant plasmid pEGHsp65 was identified by restrictive enzyme digestion and DNA sequencing. It was transfected into Hela cells by liposome, and the transfection rate of different periods were determined by laser scanning confocal microscopy so that transfection condition was optized by it. Hela cells transfected by pEGHsp65 and pEGFP-Cl respectively were collected and the total RNA was extracted. Then its cDNA was obtained by reverse transcription. That cDNA was used as the PCR template. Specific primers of Hsp65 and control of p-actin were also added into the PCR system. The cytokines rmGM-CSF rmIL-4 were added into stem cells separated aseptically from mouse bone marrow. Cultured cells in vitro were collected at 8th day and testified by both electronic microscope and optical microscope. The pEGHsp65 and pEGFP-Cl were transfected separately into dendritic cells of mice while non-transfected cells were used as normal control. Fluorescence signal wasdetected at 48 hour after transfection by laser scanning confocal microscopy. Proliferation of non-sensitized splenocytes stimulated by transfected DC was calculated by MTT.The results showed that. The recombinant plasmid pEGHsp65 was successfully constructed confirmed by restrictive enzyme digestion analysis and DNA sequencing. The pEGHsp65 was transfected into Hela cells. Fluorescence signal was observed at 24 hours after transfection and reached peak at 48 hours. The fluorescence intensity decreased at 72 hours and thereafter. Expression of Hsp65 mRNA was determined in transfected cells by RT-PCR.The transfection rate was determined by laser canning confocal microscopy. Dendritic cells were testified both by optical microscope and electronic microscope. Non-sensitized splenocytes can proliferate by the stimulation of transfected DC by MTT.We had completed the primary stage of construction of the'fusion gene of Hsp65 and enhanced green fluorescent prptein(EGFP) and preparation of dedritic cell vaccine. It lays the experimental foundation to examine whethere the new type vaccine could have the effects of therapeutic application to tuberculosis.