The Experiment Genicity And Expression Of Brucella L₇/L₁₂,Omp31 Gene And Their Fusion Gene In BCG

Brucellosis is currently the most common animal disease and widely prevalent throughout the world,which infects a variety of animals.Infection with this disease will lead to abortion,weight loss,and even death of the animal in severe cases.It is because of its serious clinical symptoms and its epidemicity,which has done serious economic harm to the domestic and foreign aquaculture industry,which has attracted extensive attention from scholars at home and abroad.Nowadays,vaccination is an internationally recognized method of preventing animal diseases.The common Brucella vaccines are mainly live attenuated vaccines,including S2,S19,M5,104M and the like.In recent years,there are more and more animals infected by Brucella.Therefore,this experiment used the L₇/L₁₂2 gene,Omp31gene and their fusion gene L₇/L₁₂-Omp31 of Brucella to construct recombinant BCG to prevent brucellosis.We are supported to make useful explorations and attempts to develop new vaccines.?1?The construction of recombinant BCGs which include rBCG-L₇/L₁₂,rBCG-Omp31,rBCG-L₇/L₁₂-Omp31The 19kDa signal peptide of BCG surface expression gene was inserted into the integrated expression vector pMV361 to construct a novel recombinant vector pMV361L which can express the foreign gene on the surface of BCG.Then,the L7/L12 gene,Omp31 gene and L7/L12-Omp31 fusion gene were inserted into the recombinant vector pMV361L by homologous recombination,and three recombinant integrated expression plasmids were constructed,which were named pMV361L-L₇/L₁₂2 and pMV361L-Omp31,pMV361L-L₇/L₁₂-Omp31.The three integrated expression plasmids were electroporated into the prepared BCG components.After acid-fast staining and bacterial liquid PCR verification,the integrated expression plasmid was successfully transferred into BCG,named as rBCG-L₇/L₁₂,rBCG-Omp31 and rBCG-L₇/L₁₂-Omp31.The three positive recombinant BCG vaccines which were identified as successfully were induced expression by thermal induction,then the total protein was extracted and detected by Western Blot.The size of the target protein was the same as the expected size and antigen specificity.?2?Immunogenicity of recombinant BCG vaccines rBCG-L₇/L₁₂,rBCG-Omp31,and rBCG-L₇/L₁₂-Omp31Three recombinant BCG vaccines were immunized intraperitoneally on day 0,day 14 and day 28,and three mice in each group were used for eyeball sampling on the 14th,28th,42th and 56th day after immunization.Serum was taken for antibody titer detection and cytokine detection.On the 56th day of immunization,spleen lymphocytes were taken for spleen lymphocyte proliferation and T lymphocyte subsets to determine the humoral and cellular immunity levels of mice.The results of antibody detectiom showed that the group of recombinant BCG could produce different levels of specific antibodies against Brucella.The results of in vitro lymphocyte proliferation assay showed that the proliferation effect of the recombinant BCG group was higher than control group,and the proliferation effect of the fusion genome was higher than the single genome.In addition,the specific stimulation with the S2 solution was significantly higher than the ConA stimulation.Therefore,recombinant BCG can induce a cellular immune response in mice.The in vitro lymphocyte proliferation test showed that the T lymphocytes of the mice presented different degrees of proliferation.The results of T lymphocyte subsets showed that the ratio of CD4⁺/CD8⁺in the recombinant BCG group was higher than the PBS group,indicating that the immune response induced by recombinant BCG was mainly CD4⁺immunization.The detection of cytokines showed that recombinant BCG could induce Th1-type and Th2-type immune responses in mice,and was dominated by Th1-type response.In addition,the recombinant BCG immune effect of the fusion gene was higher than the single gene recombinant BCG in the whole immunization process.In this experiment,the Brucella L₇/L₁₂2 and Omp31 genes were fused together and inserted into the pMV361L expression vector,which was transformed into BCG to achieve co-expression,and the recombinant BCG vaccine was constructed for immunological experiments.This will provide useful exploration and experimentation for the development of genetically engineered vaccines that are effective in preventing brucellosis.