Studies On The Construction And Expression Of Recombinant Adnovirial Vector Encoding The Mpt83 Gene Of Mycobacterium Tuberculosis

Tuberculosis is a chronic infectious disease which caused by Mycobacterium tuberculosis(MTB). MTB may invade all organs of the body,but has a predilection for the lung tissue by which causing pulmonary tuberculosis.It is estimated by WHO that 10 million new TB cases and 1.5 million deaths caused by MTB annually.China is the second most serious TB burden country in the world as approximately 400 million people were infected and over 2 million people were sick..BCG,the only available vaccine against TB,was protective against miliary tuberculosis and tuberculous meningitis in children,but has a variable protective efficacy ranging 0 to 80%in adult.Many countries took part in the new TB vaccine research,of the 200 candidates,some are in the process of animal model test,and some of them have gone into clinical phase.Tuberculosis has become more serious as the increase of drug-resistant MTB and the co-infection of HIV during recent decades,which has created an urgent need to develop new TB vaccines.It is hoped that new TB vaccine,which was safer,more effective,low cost could be developed as early as possible.In this research,recombinant adenoviral vaccine which encoding the MPT83 gene of MTB was constructed based on the Ad-Easy Adenoviral System.First of all,obtained the PMT83 gene by PCR method,then cloned this gene into the shuttle vector pAdTrack-CMV.Secondly,co-transformed the recombinant linearized shuttle plasmids pAdTrack-CMV-MPT83 and adenoviral backbone plasmids into the E.coli BJ5183,then recombinant adenoviral plasmid rAd-GFP-MPT83 was obtained through homologous recombination which occurred in E.coli B J5183.Thirdly,transfected lineafized recombinant adenoviral vector into HEK293 cell and adenovirus was packed in this cell line. Fourthly,examine the expression of MPT83 gene on mRNA and protein levels respectively in HEK293 cell.Fifthly,adenoviral vaccine was prepared by large-scale production of recombinant adenoviral particles in HEK293.Sixthly,tested the immunogenicity of recombinant adenoviral vaccine through the MTT test in vitro.Finally,vaccinated mice by recombinant adenoviral vaccine, then observed the intensity and extension of GFP expression dynamically by whole-body optical imaging technique by which observed the intensity and extension of MPT83 gene expression in vivo of mouse.The results of this research showed:(1)constructed the recombinant adenoviral vector successfully,and the transfected recombinant adenoviral vector could be packed adenovirus in HEK293 cell.Examined the MPT83 gene expression on mRNA and protein levels respectively by TR-PCR and Western-Blotting test.(2)vAd-GFP-MPT83 adenovirus had stimulated higher proliferation of human lymphocytes in vitro compare with vAd-GFP adenovirus on the same condition by MTT test which confirmed the immunogenicity of MPT83 protein.(3)observed the intensity and extension of GFP expression dynamically through whole-body optical imaging technique by which observed the intensity and extension of MPT83 gene expression in vivo of mouse,the results show that gene expression peak was occurred on fourth day of post-vaccination,considerable GPF expression was observed till half month of post-vaccination. The area of GPF expression was extended gradually after vaccination.