Construction And Preliminary Study Of Immune Effects Of B.abortus2308△wbkA And△pmm Strains

Brucellosis is a zoonotic disease caused by Brucella which is gram-negative and intracellular bacteria.Brucellosis is widely popular in China as well as in most countries of the world.The pathogen can infect a variety of livestock and people,can also infect wild animals, recently found that the bacteria can also infect marine animals. Antibiotic regimens for human brucellosis patients may last several months and are not always completely effective.While there are no vaccines for humans,several licensed live Brucella vaccines are available for livestock.These animal vaccines effect is dependent upon the host species,dose,and route of immunization.Fortunately,Vaccination is the most effective approach to prevent and control the disease in many countries.However, the practical application of vaccines is restricted because they also have a common disadvantage,such as strong virulent,caused female animals abortion,even infect humans,and can not differentiate natural infection from vaccine immunization.So we constructed deletion strains by knocking out the wbkA and pmm gene of brucella abortus strain2308,and preliminary evaluated its virulence and immune protection. Meanwhile,WBKA and PMM protein were expressed and used as coating antigen to distinguish between natural infection and vaccination of Brucella.Our aim is to screeing the vaccine candidate strain which has potential application. In this paper, our major work and the results are as follows:1. Construction and identification of Brucella abortus2308(2308) wbkA and pmm gene deletion strains (△wbkA and△pmm). The upstream and downstream sequence of the wbkA gene and pmm gene was high-fidelity amplified and fused together by fusion PCR technology. Using Bacillus subtilis as a template, we amplified SacB gene by high-fidelity PCR. The cloned upstream and downstream sequences and SacB gene were linked into the suicide vector pGEM-7zf+to construct the recombinant plasmid pGEM-7zf-AwbkA-SacB(pwS). The pGEM-7zf-△pmm-SacB (ppS) was constructed by the same way. The recombinant plasmid pwS and ppS were electroporated into Brucella abortus2308competent cells. We screened the positive colonies by ampicillin and sucrose to obtain△wbkA and△pmm gene deletion strains, then tested their genetic stability.2. Preliminary evaluation of virulence and immune effects in△wbkA and△pmm gene deletion strains:△wbkA and△pmm virulence was examined on murine macrophage RAW264.7level, using deletion mutants immuned the6-week-old mice. The virulence of different strains was evaluated by CFU counts, mice antibodies were preliminary validated by routine serological examination, the IgG antibodies and Cytokine response (IFN-y) of peripheral blood of mice were tested by ELISA, and immune protection was evaluated by inoculating Brucella2308to mices immuned after30days.3. Construction of eukaryotic expression vector pEG-wbkA and pEG-pmm and differential diagnosis:wbkA gene and pmm gene was amplified by PCR. The PCR products of wbkA gene and pmm gene confirmed by sequencing was cloned into pET-32a (+). The recombinant bacteria were transformed into E.coli BL21cells and induced by IPTG to express and then purified WBKA and PMM protein. The anti-wbkA protein antibody and anti-pmm protein antibody were detected by Western blot analysis using the purified WBKA protein and PMM protein as the first antibody to serum of mice immunizd with2308,△wbkA and□pmm as the second antibody.These results of our study showed that the Brucella2308□wbkA and□pmm had genetic stability, the△wbkA and△pmm deletion mutant strain was successfully constructed. The reverse mutation did not occur within20passages. The virulence of the△wbkA and△pmm are weaker than the parent2308, and the virulence is slightly different with the vaccine strain S19. The two mutants can induce specific humoral and cellular immunity through immunogenic and innocuity tests. The mutant strains can protect mice from infection by Brucella2308highly virulent strain, which was shown by challenge test results, and the protection force is slightly lower than S19. Western blot indicated that both△wbkA and△pmm can not induce mice to produce the corresponding anti-protein antibodies.