| Brucellosis is caused by Brucella, is one of the most important bacterial zoonosisendemic in many countries, especially in developing countries. since2000brucellosisincidence rate is rising which brucellosis is hazard to human and animals health andhinders the development of animals husbandry.In developed countries and animal husbandry undeveloped countries, massacreand bury are effective measures, but in developing countries, especially thedevelopment of animal husbandry developing countries, due to the heighter costs ofaquaculture, and the heigher infected by brucellosis, vaccination is an effective way tocontrol brucellosis that is instead of massacred and buried. While the vaccines are alsosome problems on efficiency and safety. In China Brucella abortus, B. melitensis andB. suis are very prevalence, brucellosis incidence rate is upgrade tendency. In China itis useful to utilize attenuated vaccaine S19, M5, S2to control spread of Brucella, butvaccaines themselves exist restriction: immunity proves protection with few infection.It is hard to distinguish from wild strain with vaccaine which interferes the serologictest diagnosis. So far there is no safty and efficiency vaccine to prevent human frombrucellosis.Gene deletion vaccines as one of the new genetically engineered vaccine forprevention and control brucellosis that has great prospects. Knocking pathogenvirulence-related genes, thereby reducing its virulence and activity. Type Ⅳsecretionsystem(T4SS) which is encoded by VirB operon, plays an important role onintracellula survival and persistence in the host, and VirB8is one of the importantcomponents of T4SS. Dificient VirB8gene can impact the Brucella T4SS structureand function, thus undermine its virulence and immune protection.Paper is dificient VirB8gene of B.abortus, and studying the virulence andimmunogenicity of mutant. Firstly, the VirB8upstream,downstream homology arm and sacB gene was amplified and conjuncted to cloning vectors, and then they wererestricted and conjuncted to pBK-CMV vector, constructed suicide plasmid. Thenelectronformed into brucella abortus S19competence, and screened by kan resistance,sucrose flat. The VirB8dificient with nonresistantand then screened on5%sucroseculture20generation, make sure hereditary stability. vaccine S19and VirB8genedificient infection mice, detection mice internal capable of survival, and induce micegenerate abntibodies level and body fluids IFN-γ level. Prepare vaccine and dificientimmune mice spleen cell,analysis lymphocyte subpopulations differentiationcircumstance, lymphocytes multiplication condition, detection two strains affect thecontent of spleen cell cytokine and affect generate of NO.Then, the VirB8gene wasamplified by PCR and conjuncted to cloning vectors, and then it was conjuncted toexpression vector, and expressed in E.coil. The protion was purified by HisTrapTMFF chromalography and identified by Western Blot.Experimental result:(1)Construction the suicide plasmid, success into theBrucella, screening the nonresistant dificient; compared with vaccine, mutant strainsurvival capable is descent, significantly different;(2)Vaccine and mutant strain bothinduce mice produce specificity immunity, antibodies level is no significant different,indicated mutant strain and vaccine humoral immunity level is equal; analysis themice of spleen T lymphocyte subpopulations, there is no significant different betweentwo vaccine, indicated mutant induce cellular immunity; analysis of lymphocytes ofspleen multiplication indicated mutant strain cell differentiation is less then vaccine;detection cytokine of TNF-α, IFN-γ, IL-2, IL-4, IL-10:all descend, indicatedimmunoloregulation is descend; analysis NO in the cell culture supernatant, indicatedvaccine and mutant strain has significant deviation;(3)Expressed the VirB8gene,obtained the pured protein VirB8, Molecular Weight is coincidence to theory; Finally,VirB8is a important virulence gene of Brucella S19, this gene participate in strainimmunoloregulation. |
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