Construction And Function Analysis Of The VirB12Molecular Marked Vaccine Of B.abortus Strain S19

Brucellosis, caused by brucella, is a kind of zoonotic disease. Brucella isgram-negative and intercellular bacterium. Brucellosis is a worldwide epidemic. Themost important method to control brucellosis is inoculation using vaccine, the mostpopular vaccine for cattle is S19, but it is low in protective rate and security, alsoserologic and molecular biology methods can not distinguish natural infected fromvaccine immune. These drawbacks interfere epidemiological investigation andepidemic focus research. Also there is a risk for bacterial spreading due to a potentialatavism of vaccine strain.For these reasons, in this study, we constructed a mutant uing homologousrecombination technology, which has a lower virulent than S19, with the molecularmarker which can be used to distinguish natural infected from vaccine immune.The upstream and downstream homologous sequences of virB12were amplifiedby PCR using the template of genome of S19according to the sequence fromGenebank. Meanwhile SacB gene sequences were amplified from Bacillus subtilis.The three sequences were connected to plasmid of pMD18-T respectively. Afterdouble digestion using enzymes, harvest the target fragments, connect these fragmentsto suicide plasmid of pBK-CVM, the constructed plasmid of pBK-virB12-N-C-SacBwas transformed into competent strain S19. We can select the double crossoverrecombinant through the antibiotic gene contained in the suicide plasmid and SacBgene.For analysis of the function of S19ΔvirB12, the survival capability of deletant S19ΔvirB12was tested using the infected model in macrophage RAW264.7. Usingthe mouse model to investigate the virulence and security, BALB/C mouse wereinoculated with1×106CFU of S19or S19ΔvirB12,50mouse each group. Forevaluation of the level of immunity induced by S19ΔvirB12, flow cytometry was usedto analyze CD4~+and CD8~+T lymphocytes and using ELISA to assay the level ofIFN-γ and IL-2. For determination of the protective rate, at six weeks after inoculation,the mouse were challenged with1×105CFU of B.abortus2308.Cloning VirB12gene of bovine brucella, constructing VirB12prokaryoticexpression vector using pET-28a(+) as the template, and expressing VirB12protein inE.coli BL21(DE3). The expressed product was analyzed by Western blot. Thepurified VirB12protein was used to construct a method for distinguishing the serumfrom the mouse inoculated with S19and S19ΔvirB12. Construct a method of indirectELISA for detecting bovine brucellosis using the protein of VirB12.The results show that comparing to S19, S19ΔvirB12is lower in virulence，similarity in protective rate, but higher in security.This protein has a goodimmunogenicity. Which can react to sera from S19infected mouse rather thanS19ΔvirB12. The result of IELISA indicates that using serological method candistinguish the mouse incoluated with S19and S19ΔvirB12. The constructed mutantof S19ΔvirB12is a potential vaccine with the characters of low virlence, high securityand can be distinguished by serological method.