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TB (Tuberculosis, TB) is Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) infection caused by chronic diseases, MTB may invade various organs of human body, but mainly affects the lungs, called pulmonary tuberculosis. According to the World Health Organization (WHO) statistics, every year there are 10 million new cases and 1.5 million died of tuberculosis. Highest in the world of high TB ??burden countries second place, the existing MTB infection has reached 400 million, infectious TB patients reached 2 million. BCG (BCG) as the only vaccine for the prevention of tuberculosis, for children with severe tuberculosis, such as miliary tuberculosis and tuberculous meningitis has a preventive effect, but for the prevention of adult TB is not satisfactory, ranging from 0-80% protection rate . Currently, many countries are studying new TB vaccine, has more than 200 kinds of candidate vaccines, some of which is in progress in animal models, and some have entered clinical trials. In recent decades, with the increase in drug-resistant MTB and HIV co-infection and other problems, increased the severity of tuberculosis. Therefore, the development of new effective TB vaccine is imminent, we hope to develop a more secure, efficient, low cost, and can be widely used in all kinds of people of the new TB vaccine. In this paper, the widely used Ad-Easy adenovirus packaging system was constructed based on Mycobacterium tuberculosis MPT83 recombinant vaccine. First, PCR for gene of Mycobacterium tuberculosis MPT83, and then the shuttle vector recombinant MPT83 pAdTrack-CMV; through chemical conversion method linearized recombinant shuttle plasmid (pAdTrack-CMV-MPT83) and viral backbone plasmid (pAdEasy-1) were transformed into E. coli BJ5183, recombinant shuttle plasmid and viral backbone plasmid formation of homologous recombination in BJ5183 adenovirus plasmid (rAd-GFP-MPT83); linearized recombinant plasmid was transfected into HEK293 cells and in HEK293 cells packaged into virus; mRNA and protein levels of target gene expression detected; lot adenovirus was amplified in vitro by the MTT preliminary experiments verify the immunogenicity of recombinant adenovirus; vivo via recombinant adenovirus immunized mice, the overall observed fluorescence imaging reporter gene green fluorescent protein (GFP) expression in mice and distribution of indirect tracking target gene expression in mice. The results showed that: (1) Mycobacterium tuberculosis MPT83 successfully constructed recombinant adenovirus vector to construct the recombinant adenovirus vector in HEK293 cells successfully packaged adenovirus. By RT-PCR and Western-Blotting experiments, mRNA and protein levels were detected on the show that Mycobacterium tuberculosis MPT83 gene expression in HEK293 cells. (2) MTT experiments show: vAd-GFP-MPT83 liquid ratio vAd-GFP virus virus solution has a stronger ability to stimulate lymphocyte proliferation in vitro, confirming the protein has a strong ability to stimulate lymphocyte proliferation, i.e. the immune immunogenicity. (3) in vivo experiments in mice showed that: vAd-GFP-MPT83 virus of mice immunized GFP reporter gene after vaccination 4d peaked until after two weeks there is a strong expression, while the scope of the expression by the The original site of injection to the periphery spread.
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