Study On The Preparation And Immune Effect Of New Mycobacterium Tuberculosis Vaccine

Part ? Construction and immunogenicity investigation of Hsp65-Ag85B/Hsp65-ESAT6 fusion gene DNA vaccine strain of Mycobacterium Tuberculosis Objective Tuberculosis(TB),a chronic respiratory infectious disease caused by Mycobacterium tuberculosis(MTB)infection,is listed as one of the major infectious diseases in China and seriously endangers the health of people in developing countries.The purpose of this study is to construct and identify candidate vaccine strains of Mycobacterium tuberculosis Hsp65-Ag85 B and Hsp65-ESAT6 DNA,and to initially evaluate their immunogenicity.Methods The PVAX1-Hsp65-Ag85 B and PVAX1-Hsp65-ESAT6 plasmids were constructed by gene synthesis,doubleenzyme digestion and ligation,and the correctness of the sequences were identified by PCR,agarose gel electrophoresis and sequencing.Results Western blot showed after recombinant plasmid transfection into 293 T cells for 24 and 48 hours,there were clear and specific target protein bands;the results of mouse immunoassay showed that DNA vaccine could induce high level of TNF-?,which was responsible for Th-type immune response.Conclusion The synthesized stable DNA vaccine in vitro has good immunogenicity,which lays a foundation for the improvement of DNA vaccine research and provides new ideas for the development of tuberculosis vaccine.Part ? Construction and screening of ure C gene deletion strain of BCGObjective The Cre/Lox P homologous recombination method was used for the genetic manipulation of BCG vaccine,and the nitrogen source gene ure C in BCG vaccine was deleted,which provided the basis for the subsequent addition of effective MTB immune antigen in this deletion strain.Methods The p SL002 plasmid containing recombinant enzyme was transferred to BCG vaccine by Cre/Lox P homologous recombination method,and the single colony was grown to perform the sensing state.Used spe? and Nsi? double enzyme about this early laboratory building contains ure C gene homologous arm and recombinant plasmid p SL001 GFP fluorescent protein,for purpose,and its telegramed to the above contains p SL002 plasmid of BCG,pick monoclonal after coated board,through the microbial PCR identification,screening the BCG vaccine in double exchange missing ure C gene fragments and strains,to obtain the BCG vaccine ure C gene deletion strains.Results M.calmette-guerin ure C gene deletion strain was successfully screened with correct morphology and stable passage.Conclusion The Cre/Lox P homologous recombination method was used to carry out genetic manipulation in BCG vaccine,and the gene of BCG vaccine could be knocked out seamlessly,and the obtained mutant could be passed on stably.The application of Cre/Lox P homologous recombination system can greatly improve the MTB recombination efficiency and shorten the recombination time,which provides convenience for the study of recombinant BCG vaccine.