Construction Of Recombinant Adenovirus With Ag85a Gene Of M.bovis

As is well-known, zoonoses have been the key factor of threating the human health,hindering the development of animal husbandry. Bovine tuberculosis is a serious infectiousdisease all around the world. Bovine tuberculosis is caused by Mycobacterium bovis,and haswidespread hosts. Livestock and a variety of wild animals can be pathogenic. For a long time,human and cattle are very closely intertwined in a variety of ways such as cattle breeding, cattleproducts, and this also results in the spread of bovine tuberculosis to humans, so it is a seriousimpact on human health. Since the successful listing of BCG, TB has been controled to a certainextent. But in recent years with the protective effect of BCG declining, the cases of immunedeficiency disorders increasing and the rapid development of world trade, TB strikes againwith a substantial rate. Human search for new vaccines continually to prevent and controltuberculosis, but we have not successfully developed more effective ones than BCG. Therefore,the development of more secure and efficient vaccines against tuberculosis has a very importantsignificance.Adenoviral vector has been the hot topic of studying viral vectors.Because of its high safety,wide host, efficient stable expression of exogenous genes, it is widely used in many fields suchas gene therapy and vaccination currently. With the further study on adenoviral vector,researchers make more and more efficient and safe adenoviral vectors gradually come out byconstantly modifying adenovirus vectors. Especially RAPAd adenovirus expression system,itsoperation is simple and fast.This system can efficiently obtian replication-defectiveadenovirus.Therefore the expression system is usually used as one of the preferrde tools to buildand obtain recombinant adenovirus.In this study,the ag85a gene of HindⅢ locus mutation was amplified by SOE PCR,thenthe ag85a gene was connected to the pMD-18-T vector to construct a reconbinant plasmidclones pMD-85a.The recombinant plasmid pMD-85a and replication-defective adenovirusshuttle vector pacAd5CMV were digested by restriction end onuclease of HindⅢ andEcoRⅠ,then the ag85a gene was inserted into the shuttle vecctor pacAd5CMV to construct therecombinant adenovirus shuttle plasmid pacAd5CMV-ag85a.Then co-transfect293AD cellswith the recombinant shuttle plasmid and backbone plasmid which are linearized with arestriction endonuclease Pac Ⅰ.The recombinant shuttle plasmid and backbone plasmidcomplete the homologous recombination in the cells in order to get the replication-deficientrecombinant adenovirus(human adenovirus serotype) pacAd5-ag85a containing the ag85a gene.The pacAd5CMV-GFP Control Vector is used the same method to construct recombinant virus pacAd5CMV-GFP with GFP. The recombinant virus pacAd5CMV-GFP with GFP is a contrastto preliminarily identify the successful construction of the recombinant virus. We confirm thatthe recombinant adenovirus pacAd5-ag85a can express Ag85A antigen in293AD cells by theindirect IF staining.All of these provide a theoretical basis for the study of pacAd5-ag85a as anew tuberculosis vaccine.