Study Of Immune Effect Based On BCG Priming And Mycobacterium Tuberculosis DNA Vaccine Boosting Strategy In Mice

Tuberculosis(TB) is a chronic respiratory infectious disease that has afflicted humans for thousands of years. For more than 80 years, the bacillus Galmette-Guérin(BCG) vaccine has been the only licensed TB vaccine given to humans covering 86% of the world population. However, despite the use of BCG, TB remains a global epidemic with one-third of the world population being infected and per year morbidity of 8 million new cases and 2 million deaths were happened. Unluckily, incidence of TB is being accelerated by the spread of HIV/AIDS and the increasing Mycobacterium tuberculosis of multiple resistance to drug. Many studies suggests that BCG vaccination is protective against childhood meningeal tuberculosis and systemic forms of the disease. However, protective efficacy is variable against adult pulmonary disease (ranging from 0-80%) and the immunization effect of BCG wanes with time. Furthermore, BCG vaccine may cause severe complications in immunocompromised hosts. Thus, there is an urgent need for developing more safe, effective TB vaccines, new delivery methods and strategy that are able to confer potent protection. According to this thinking, we make our research on TB DNA vaccine in two parts as below.1. Design, construction and identification of eukaryotic co-expression vectors in view of TB DNA vaccineObjective: To Design, construct and identify two kind of eukaryotic co-expression recombinant DNA vaccines, one of which contains mouse interleukin 21(IL-21) and tuberculosis antigen 85A(Ag85A) and another contains mouse Granulocyte macrophage colony stimulating factor(GM-CSF) and Ag85A.Methods: The gene of IL-21 was amplified from recombinant plasmid pcDNA3.1-mIL21 by PCR and cloned directly into the plasmid pRSC, and form recombinant plasmid pRSC-IL21. The gene of Ag85A was amplified from the recombinant plasmid pIRES-Ag85A by PCR and cloned directly into the recombinant pRSC-IL21 and recombinant pRSC-GMCSF respectively again, and formed co-expression DNA vaccine pRSC-mIL21-Ag85A and pRSC-mGMCSF-Ag85A separately finally.Results: The eukaryotic co-expression DNA vaccine plasmid containing IL-21 and Ag85A or the DNA vaccine containing GMCSF and Ag85A had been constructed correctly by the identification of endonuclease digestion and DNA sequencing. The protein expression of co-expression DNA vaccine had been demonstrated correctly by RT-PCR method.Conclutions: The co-expression DNA vaccines of pRSC-mIL21-Ag85A and pRSC-mGMCSF-Ag85A have been constructed successfully. This study provides the possibility of further research on the development of new type anti-tuberculosis vaccines.2. Study of immune strategy and efficiency based on BCG priming and...