omp25c基因对马耳他布鲁菌疫苗株M5的毒力及免疫保护性的影响

The Effect of omp25c Gene on Virulence and Protective Efficacy of M5 Strain Against Brucella melitensis in Mice

目的:初步评价马耳他布鲁菌M5疫苗株omp25c基因对其毒力及免疫保护性的影响。方法:利用同源重组的方法,用卡那霉素抗性基因替换M5的omp25c(BMEI1829)基因,得到缺失突变株M5Δomp25c;分别用M5Δomp25c和M5免疫小鼠,在免疫后不同时间点处死小鼠,通过脾脏细菌计数分析缺失突变株在小鼠体内的毒力,通过检测IgG和IFN-γ的水平分析缺失突变株在小鼠体内诱导的体液和细胞免疫应答能力,通过攻毒实验评价突变株的免疫保护效果。结果:与M5株相比,M5Δomp25c在小鼠脾脏内的存活时间较短,在第4周时未能检出;M5Δomp25c免疫小鼠诱导产生的IgG水平在第4周达到最高,第6周开始下降;M5Δomp25c免疫小鼠诱导分泌的IFN-γ水平在第4周达到最高为790pg/mL,第6周时浓度降至530pg/mL,整体趋势显著低于阳性对照组;接种了M5Δomp25c的小鼠用布鲁菌强毒株16M攻毒后,免疫保护效果也下降。结论:缺失omp25c的突变株毒力减弱,诱导的体液和细胞免疫水平及免疫保护效果下降,说明omp25c基因是马耳他布鲁菌M5疫苗株的毒力相关基因,对疫苗株M5的免疫应答和免疫保护效果有一定的影响。

Objective： To evaluate the effect of omp25c gene on virulence and protection of Brucella melitensis M5 strain. Methods： M5 was genetically modified by homologous recombination, the omp25c gene was replaced by kanamycin resistance gene, generating MSAomp25c. BALB/c mice were infected with M5 and M5Aomp25c respectively. The virulence was assessed by the mean CFU/spleen at every time point. IgG and IFN-γ level was detected to analyze the humoral and cellular immune response. The protective efficacy was evaluated by challenge experiment. Results： BALB/c mice infected with MS△omp25c had significant lower mean CFU/spleen counts from one to four weeks post-infection and the mutant was cleared from the spleens at four weeks post-infection. IgG level induced by MSAomp25e was highest at four weeks post-infection and dropped at six weeks post-infection. IFN-γ level induced by MS△omp25c increased to 790 pg/mL at four weeks post-infection and dropped to 530 pg/mL at six weeks post-infection, which showed significant lower than that induced by M5 strain. Challenged mice infected by the mutant resulted lower protection against virulent strain 16M than vaccine strain M5. Conclusion： The mutant showed lower virulence, and inducing decreased humoral and cellular response and protection compared to vaccine strain M5, suggesting that omp25c gene significantly affects immune response and protective efficacy of strain M5.