布鲁氏菌M28强毒株O抗原聚合酶基因缺失株的构建及其毒力评价  

Construction and virulence evaluation of O-antigen polymerase deficient Brucella melitensis M28 strain

作　　者：赵云[1] 胡森[1] 乔祖建[1] 刘文兴[1] 覃晓琳[2] 步志高[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部兽医公共卫生重点开放实验室,黑龙江哈尔滨150001 [2]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《中国预防兽医学报》2013年第3期197-201,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31001068)

摘　　要：为构建布鲁氏菌O抗原聚合酶缺失突变株,本研究以布鲁氏菌M28株为亲本株,利用同源重组方法,以编码O抗原聚合酶靶基因M28_B0107基因ORF外侧序列作为同源臂构建重组质粒pSP-B0107-K,将其电转化至M28感受态细胞中,以卡那霉素抗性基因(Kanr)作为标记,筛选缺失突变株(M28-ΔB0107)。M28-ΔB0107经过20余代传代培养,Kanr表达稳定。将M28-ΔB0107与M28以106cfu剂量腹腔接种小鼠,进行体内致病性试验。结果显示,M28-ΔB0107感染组小鼠脾脏荷菌量显著低于亲本M28株感染组,感染6周时,前者低于后者近10倍(p<0.05);而且M28-ΔB0107感染组脾脏重量也显著低于M28强毒株组(p<0.05);小鼠腹腔巨噬细胞系(RAW264.7)感染能力试验结果表明,突变株与亲本株无明显差异(p>0.05)。To construct an O-antigen polymerase deficient Brucella melitensis wild strain M28 （M28-△B0107） and evaluate the virulence of the M28-△B0107, we replaced the O-antigen polymerase encoding gene B0107 in the B.melitensis M28 with kanamycin resistance gene based on the method of homologous recombination and tested the pathogenicity of the M28-△B0107 in mice and the replication in cell culture. The pathogenicity result showed that the M28-△B0107 was still able to cause the mouse spleen swollen, but twice smaller than that in the mice infected with the parental bacteria, in addition, the bacteria load in the spleen was ten times lower than that in the mice infected with the parental bacteria at 6 week post infection. However, the bacterial counts displayed that the survival rate of M28-△B0107 had no difference to the parental strain in the murine macrophage cell line RAW264.7. These results indicate O-antigen polymerase is one of the virulent factors of B.melitensis.

关 键 词：布鲁氏菌 O抗原聚合酶 B0107 

分 类 号：S852.61[农业科学—基础兽医学]