| Brucellosis is a serious disease affecting both human and animals; the disease isspreading all over the world. Attenuated live vaccine for livestock has been widely usedfor many years to prevent the disease; however it has the risk of occasional bacterialreplication in inoculated animal bodies. The genetically engineered vaccines have beentried to overcome the problem, but they failed to induce strong mucosal immunity againstBrucella. Transgenic plant vaccines showed promise in several aspects. The vaccines inthis category have been demonstrated to be able to stimulate mucosal immunity; inaddition, they are edible so that do not require injection, and are safer than both livevaccine and genetically engineered vaccines.For development plant edible vaccines, the fusion Brucella gene rOmp3148-74-BLS wastransferred into alfalfa Medicago sativa Zhongmu No.1mediated by Agrobacterium in thisstudy.Towards that end, gene rOmp3148-74-BLS from our lab collection was amplified usingLIC (ligation independent cloning) primers, and then the gene was inserted into vectorpJG045after digested with Apa I. The recombinant vector pJG-OB was used to transformAgrobacterium AGL0by tri-parental hybrid method and positive transformants werescreened with colony-PCR; sequencing confirmed Agrobacterium carrying pJG-OB wasused to infect explants of alfalfa. The cotyledon explants or hypocoty explants to beinfected were prepared from disinfected alfalfa seedling. The explants were co-incubatedwith the vector carrying Agrobacterium, PCR was used to identify the transgenic explantsfrom which transformed calli were obtained. The positive calli were identified by PCR;after stimulating the calli for growth of roots, transgenic alfalfa was obtained from theroots.The gene rOmp3148-74-BLS contained Brucella OMP31antigenic epitope spanningamino acids48-74and the lumazine synthase, an enhancer for antigens; the fusion protein has been shown by coworkers to be strongly antigenic. Thus, this experiment has laid basisfor the development of Brucella transgenic plant vaccine in the future. |
