羊布鲁菌16MUGPase基因缺失株的构建及其特性分析  

Construction and characteristics of UGPase gene-deleted mutant of Brucella melitensis 16M

作　　者：卜昭阳[1,2] 杨艳玲[3] 郎需龙[2] 钱晶[4] 李诗雨[2] 李争[1] 卢晓冉 沙洲[1] 王莉[1] 王兴龙[2] 

机构地区：[1]吉林农业大学动物科技学院,吉林长春130062 [2]军事医学科学院军事兽医研究所,吉林长春130062 [3]中国农业科学院特产研究所,吉林长春130114 [4]吉林大学动物医学学院,吉林长春130062

出　　处：《中国生物制品学杂志》2017年第7期690-694,共5页Chinese Journal of Biologicals

摘　　要：目的构建羊布鲁菌16M株的尿苷三磷酸-葡萄糖-1-磷酸-尿苷酰基转移酶(UTP-glucose-1-phosphate-uridylyltransferase,简称UGPase)基因缺失候选疫苗株,并对其侵染宿主巨噬细胞的能力及毒力进行初步分析。方法采用PCR技术对羊布鲁菌的UGPase基因上、下游同源臂进行克隆,利用基因同源重组技术构建自杀质粒,经两步筛选法对重组菌株筛选后,进行巨噬细胞黏附侵袭试验和小鼠体内毒力试验。结果自杀质粒p BK-CMV-Sac B-△UGP构建成功,经双向筛选获得了羊布鲁菌UGPase基因缺失株16M△UGP。16M△UGP缺失株侵入巨噬细胞的CFU数低于16M强毒株,但高于M5疫苗株;小鼠攻毒14 d后,其平均脾指数为(6.55±0.27)g,平均克脾菌数为1.64×104CFU/g,均低于16M强毒株。结论成功构建了羊布鲁菌16M株的UGPase基因缺失株,并证实了UGPase基因的缺失对16M株毒力具有一定影响。Objective To construct the UTP-glucose-1-phosphate-nridyly-1 transferase （UGPase） gene-deleted mutant of Brucella melitensis 16M vaccine candidate, and preliminary analyze its ability to infect the host macrophages and virulence. Methods The upstream and downstream homologous arms of Brucella UGPase gene were cloned by PCR, based on which recombinant suicide plasmid was constructed by gene homologous recombination technology. Recom- binant strains were screened by the two step screening and subjected to macrophage adhesion test and virulence test in vivo in mice. Results Recombinant suicide plasmid pBK-CMV-SacB-A UGP was successfully constructed, and UGPase gene-deleted mutant 16M A UGP was obtained by dual screening. Macrophage adhesion test showed that the CFU of 16MAUGP in macrophages was less than that of B. melitensis 16M but was more than that of B. nvelitensis 5M. The mean spleen index of mice 14 d after challenge was （6. 55 ± 0. 27） g, while the mean spleen index per gram was 1.64 × 10^4 CFU / g, both of which were lower than those of B. melitensis 16M. Conclusion The UGPase gene-deleted mutant of B. melitensis 16M was successfully constructed, which proved that the deletion of UGPase gene showed a certain effect on the virulence of B. melitensis 16M.

关 键 词：羊布鲁菌 UGPase基因缺失株 毒力 

分 类 号：R378.52[医药卫生—病原生物学]