布鲁氏菌omp25重组BCG的构建及其疫苗作用检测

发布时间：2018-10-24 11:14

【摘要】：目的构建分泌性表达布鲁氏菌外膜蛋白OMP25基因重组卡介苗(rBCG-omp25),并免疫BALB/c小鼠,观察其免疫作用。方法利用分子生物学技术构建重组穿梭分泌载体pMV261-Ag85B-omp25,电穿孔技术导入卡介苗(BCG)。通过抗生素筛选、重组卡介苗基因组PCR扩增、测序,以及Western blot对重组卡介苗进行鉴定。分别用rBCG-omp25、BCG腹腔接种BALB/c小鼠,于免疫后第10、20、30、40和50d称重,尾部采血,用表达纯化的融合蛋白OMP25-32a检测免疫小鼠抗体的生成情况,用流式细胞仪(FCM)检测CD4+、CD8+T淋巴细胞百分率,并计算CD4+/CD8+T细胞比值。制作小鼠肝组织切片,HE染色观察病理学变化。结果重组卡介苗rBCG-omp25含有Ag85Bomp25序列,大小为745bp;Western blot表明rBCG-omp25可分泌表达布鲁氏菌OMP25。rBCG-omp25免疫小鼠后第20d,用Western blot检测到布鲁氏菌OMP25特异性抗体;rBCG-omp25免疫组和BCG免疫组的CD4+、CD8+T细胞百分率分别为38.68%、11.32%和、48.44%、14.01%,PBS组为33.24%、9.81%,差异有统计学意义(P0.05)。免疫后50d内rBCG-omp25组、BCG组与PBS组小鼠体重差异无统计学意义(P0.05);各实验组小鼠肝组织均未见明显病理改变。结论构建的rBCG-omp25能够表达特异性蛋白OMP25。该蛋白能刺激小鼠产生特异性抗体,能刺激小鼠外周血CD4+、CD8+T淋巴细胞增殖。rBCG-omp25毒力弱,可作为预防布鲁氏菌病的疫苗候选株之一。
[Abstract]:Objective to construct recombinant BCG vaccine (rBCG-omp25) expressing OMP25 gene of Brucella spp. Methods Recombinant shuttle secretory vector pMV261-Ag85B-omp25, electroporation technique was used to construct recombinant shuttle secretory vector pMV261-Ag85B-omp25, into BCG (BCG). By molecular biology technique. The recombinant BCG vaccine was identified by antibiotic screening, PCR amplification, sequencing and Western blot. BALB/c mice were inoculated intraperitoneally with rBCG-omp25,BCG and weighed on the 10th day after immunization. Blood was collected from the tail. The antibody production was detected by OMP25-32a, and the percentage of CD4 and CD8 T lymphocytes was detected by flow cytometry (FCM). The ratio of CD4 / CD8 T cells was calculated. The liver tissue sections of mice were made and the pathological changes were observed by HE staining. Results the recombinant BCG rBCG-omp25 contained Ag85Bomp25 sequence. The specific antibody to brucella OMP25 was detected by Western blot on the 20th day after immunization with OMP25.rBCG-omp25. The percentage of CD4 and CD8 T cells in rBCG-omp25 immunized group and BCG immunized group were 38.68% and 11.32%, and 48.44 ~ 14.01%, 33.240.81%, respectively. The difference was statistically significant (P0.05). There was no significant difference in body weight between rBCG-omp25 group, BCG group and PBS group within 50 days after immunization (P0.05). Conclusion the constructed rBCG-omp25 can express the specific protein OMP25.. The protein can stimulate the production of specific antibodies and the proliferation of CD4 and CD8 T lymphocytes in peripheral blood of mice. RBCG-omp25 is weak in virulence and can be used as one of the vaccine candidate strains to prevent brucellosis.
【作者单位】：石河子大学医学院病原生物学与免疫学教研室;阿克苏地区天山肉用种羊基地畜牧兽医站;新疆石河子市人民医院;石河子大学动物科技学院;
【基金】：国家科技支撑计划(No.2013BAI05B05) 国际科技合作项目(No.2013DFA32380) 国家自然科学基金项目(No.31060334)
【分类号】：R392.1

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