Construction Of New Types Of Vaccines Of Brucella And Evaluation Of Their Immunogenicity

Brucellosis is a zoonotic disease caused by a small intracellular gram-negative bacterium which is pathogenic for humans as well as for many species of animals. This infection induces abortions, in livestock animals as well as undulant fever and osteomyelitis in humans leading to severe economic losses. For example, United States Department of Agriculture sources estimated that in the 1990s, on average about US$150 million were spent each year in the US. There are six species Brucella. Abortus, B. suis, B. melitensis ,B. canis, B.ovis and B.neotomae. Control of the spread of the disease in domesticated animals is the use of vaccines for whole-herd vaccination programs. But the killed vaccines and live vaccines have been limited because of their unacceptable production costs, poor protection and serological problem which interfere with the diagnosis of field infection. And the recombinant subunit vaccine has many advantages: first, it's easy to make and does not cost much money; second, it's safe and easy to master the quantity; finally, it can be used through all kinds of delivery system. DNA vaccine also attracted the eyes of many scientists, after injection into the muscles, it can express the protein for a long time, and the protein degrades into peptides and associates with major histocompatibility complex (MHC) class I molecules. These peptide/MHC complexes stimulate cytolytic T cells, induce cell mediated immune response(CMI) to control the infection. L7/L12 ribosome protein is the best known T-cell antigen .Studies have confirmed that recombinant L7/L12 protein could specifically stimulate mononulclear cells from B.abortus reactive animals and upregulate the transcription and expression of IFN-γand L7/L12 DNA vaccine also engendered significant protection.The OMP16 outer membrane lipoprotein exists in all the six species of Brucella. It has been proved to be a protective antigen too; The sequence of 17.3kDa protein shows homology with that of lumazine synthase, indicates that the two protein may share the same characters, and the lumazine synthase is an enzyme with lumazine synthase activity which can induce T-cell activity. Because of this reason, the 17.3kDa protein should be a protective protein. Using B epitope soft assay suggests that the initialfactor 3 has immungenicity, although we can not find anti-IF3 antibody in the sera of Brucella infected animal, probably the protein can elicit cytokines and co-react with other antibody.We selected these 4 proteins, constructed recombinant eukaryotic protein and expressed the corresponding recombinant protein at the same time, immunized animals and evaluated their capability in inducing humoral immunity and celluar immunity. The whole genome was isolated from the B.melitensis, and amplicated the corresponding gene using PCR techniques. The genes were inserted into pET32a(+) prokaryotic expression vector and then expressed the proteins;genes were cloned into pCDNA3.1 too, transfected the recombinant plasmids and identified proteins expression using Western blot analyses and immunochemistry. Injection of these recombinant plasmid DNA into BALB/c mice via an intramuscular route together with plasmid DNA carrying GM-CSF gene and recombinant proteins mixed with Freund's adjuvant via an intraperitoneal injection elicited both humoral and cellular immune responses. Experimental animals exhibited specific antibodies that were detectable 15 days after the first injection and increased steadily, the IgG2a-specific antibody titers of DNA groups were higher than IgG1-specific antibody titers, and the recombinant protein groups were contrary. The DNA vaccines elicited a T-cell-proliferative responses as well as IFN-γ production upon restimulation with the specific antigens using ELISPOT assay; the ratio of CD4+/CD8+ were lower than that of the control mice. Furthermore, immunoglobulin G isotype analysis indicated that the ratio of IgG1 and IgG2a in protein vaccinated group is higher than 1 and that of DNA vaccine groups lower than 1,suggesting the i...