| Bovine tuberculosis is a zoonosis which threats human health and hinders the development of animal husbandry.The infection of bovine tuberculosis is global, and bovine tuberculosis is a chronic wasting diseases caused mainly by Mycobacterium bovis, whose onset is not distinctly seasonal.Its host is very extensive and cattle is most susceptible, at the same time people, pigs, poultry and wild animals can also be ill.The contact of cattle and human is very close on cattle breeding and consumption of cattle products and many other sides, which lead to people infected with bovine tuberculosis and threat human health seriously.Today, Bacillus Calmette-Guerin(BCG) is still the only available vaccine against tuberculosis.However, due to the immunological protection of BCG decreased, the cases of immunodeficiency disease increasing continuously, BCG cannot be applied in immunocompromised individuals and environmental mycobacteria often leading to immune failure of BCG. Therefore, all the countries in the world are committed to the development of improved BCG or more safe and efficient novel TB vaccines.Ag85 complexes are early extracellular secreting antigens secreted by Mycobacterium tuberculosis.Research shows the Ag85 complexes are mainly composed of Ag85 A, Ag85 B and Ag85 C. Ag85 A as the higher secretion rate component among them, can cause cellular immune response significantly, so it is a ideal antigen of Mycobacterium tuberculosis subunit vaccine. The DNA vaccine structured of ag85 a can induce anti-tuberculosis immune response, it is a candidate gene of DNA vaccine. In recent years, the excellent immunogenicity of Ag85 A has received substantial attention, and a variety of tuberculosis vaccines carrying Ag85 a gene have entered clinical trials and have obtained ideal results.Lactic acid bacteria(LAB) belonging to gram-positive bacterium are a group of industrial strains which are widely applied in the food. Lactic acid bacteria is closely related to human beings, some of which are normal flora in the body and have a beneficial effect on the body.Lactic acid bacteria play an important role in the prevention of food corruption, immunity boost and the improvement of the flavor. With the further in-depth study under molecular structure, gene composition and unique properties of lactic acid bacteria, applications of lactic acid bacteria are no longer confined to food industry, and as host bacteria it has made significant progress in the construction of expression vector and the expression of heterologous protein. As some lactic acid bacteria are normal intestinal flora of human and animals, so lactic acid bacteria that are food grade security as vector of presenting antigen can be made into oral vaccine which has obtained good immune effect through the reaction with intestinal mucosa.In this study, ag85 a gene of Mycobacterium bovis was amplified by PCR technique, then it is connected with the pMD18-T sample vector to construct the recombinant plasmid pMD18-T(S)-ag85 a.The recombinant plasmid pMD18-T(S)-ag85 a and expression vector pNZ8149 of lactic acid bacteria were digested with restriction endonucleases KpnⅠand NcoⅠ, then Ag85 A gene was inserted into the expression vector pNZ8149 to construct the recombinant expression plasmid pNZ8149-ag85 a.The recombinant expression plasmid pNZ8149-ag85 a was transformed into host bacteria NZ3900 with electrotransformation to construct the recombinant lactoeoeeus lactis with Ag85 a gene.The recombinant lactoeoeeus lactis was induced by 5ng/mL Nisin, then the SDS-PAGE analysis showed that the protein molecular weight is about 31 kDa.Mice were fed with the recombinant lactoeoeeus lactis, then we study on and analyze the immune response of mice.It may lay a foundation for researching the recombinant lactoeoeeus lactis pNZ8149-ag85a/NZ390 as a new vaccine for bovine tuberculosis. |
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