Construction And Immunological Study Of Brucella A19-?Omp16 Gene Deletion Vaccine Strain

Brucellosis is a zoonotic infectious disease caused by Brucella infection in animals or humans.It can make patients with joint pain,muscle aches,long-term fever,and sweating.Brucellosis in humans is usually transmitted by animals.At present,the prevention and control of brucellosis in animals is mainly based on prevention,and positive animals are eliminated.Vaccination is an effective measure to prevent brucellosis.At present,the brucellosis vaccines on the market in China are all attenuated vaccines.The attenuated vaccines against brucellosis can still be used by staff in livestock farms.Or the laboratory operator caused the infection,and the vaccine strain still has a certain degree of virulence.If the female animal is vaccinated against Brucella attenuated during pregnancy,it will also cause its abortion,and the current serological testing methods cannot effectively distinguish the animal vaccine strain Antibody response caused by immune and wild strain infection.Therefore,the development of a safe,reliable and easily identifiable brucellosis vaccine has become the current focus of research.In this experiment,the Omp16(BMEI0340)gene was used as the research target.First,primers were designed for its ORF region and PCR amplified.The primers were ligated to the p ET-28 a vector and transferred to BL21 competent cells.A prokaryotic expression strain of Omp16 was constructed.The Omp16 protein was purified after SDS-PAGE and western blot verification and was used for subsequent use.The primers were designed to PCR-amplify the upstream and downstream sequences of the Omp16 gene(BMEI0340).The kana resistance sequence fragment was selected on the y TREX-Sac B plasmid gene sequence to design primers for amplification.The three segments of genes were spliced by fusion PCR and ligated to the p MD18-T vector was transformed into the B.abrotus A19 vaccine strain by electroporation and homologous recombination with its genome.The kana resistance gene was substituted for the Omp16 gene to construct an A19-?Omp16 deletion mutant.The A19-?Omp16 deletion mutant was passaged for 24 consecutive generations and tested by PCR,and no reverse mutation was found.The A19-?Omp16 deletion mutant strain was placed in different external environments to test the stress ability.It was found that after the deletion of the Omp16 gene,the bacteria's tolerance to the external environment was generally weaker than that of B.abrotus A19 due to the change in membrane structure.Parent strain.A19-?Omp16 deletion mutant and B.abrotus A19 parental strain were infected with Raw264.7 mouse macrophages and six-week-old female Balb / c mice,respectively,and compared their viability in macrophages and mice and Toxicity to mice.The results of macrophage challenge experiments showed that the content of A19-?Omp16 deletion mutants was less than that of B.abrotus A19 parent strains at different time points after challenge.The results of mouse infection experiments showed that the spleen index and spleen load of mice inoculated with A19-?Omp16 deletion mutant were lower than those inoculated with B.abrotus A19 parent strain at different time points.The mice were immunized with the A19-?Omp16 deletion mutant and the B.abrotus A19 parental strain,respectively,and then challenged with the B.abrotus 2308 virulent strain to test the immune protection of the deleted strain on the mice.The spleen index and spleen load of mice immunized with the A19-?Omp16 deletion mutant were slightly lower than those immunized with the B.abrotus A19 parent strain two weeks after challenge,but the differences were not significant.The Omp16 protein purified before was loaded and tested by western blot to verify the antibodies in the serum.The serum of mice inoculated with the B.abrotus A19 parent strain and the mouse serum inoculated with the A19-?Omp16 deletion mutant were used as primary antibodies and labeled with HRP.The goat anti-mouse serum was a secondary antibody,and the results showed that no bands appeared on the NC membrane of the mouse serum inoculated with the A19-?Omp16 deletion mutant strain as the primary antibody,while those inoculated with the B.abrotus A19 parent strain A band appeared on the NC membrane of the mouse serum as the primary antibody,indicating that the serum of the mouse immunized with the A19-?Omp16 deletion mutant did not contain an antibody to the Omp16 protein.The above experimental results show that this experiment successfully constructed a stable inheritable A19-?Omp16 deletion mutant,and after the Omp16 gene was deleted,the bacteria's tolerance to the external environment was weakened,and survival in macrophages and mice The ability is reduced to a certain extent,and the virulence is weakened to become safer,and it can provide similar immune protection to the parental strain.The immunized mice do not produce antibodies against Omp16 protein,which can be used for vaccine immunization The distinction between viral infections is a promising candidate strain of brucellosis.