布鲁氏菌043强毒株BR0524基因缺失突变株的构建与毒力的初步评价

为研究BR0524基因对羊种布鲁氏菌043毒力的影响,本研究利用同源重组的原理,以BR0524基因外侧的同源臂序列构建重组质粒,将其电转化至布鲁氏菌043感受态细胞,通过卡那抗性筛选和检测引物鉴定之后进而获得羊种布鲁氏菌043-ΔBR0524缺失突变株,且该缺失突变株在15代内未发生回复性突变,遗传性稳定。将羊种布鲁氏菌043-ΔBR0524缺失突变株、羊种布鲁氏菌043和M5以106 CFU剂量腹腔接种小鼠,进行体内感染试验,并以布鲁氏菌与小鼠巨噬细胞为100∶1的感染比例侵染小鼠巨噬细胞RAW264.7。结果表明:与羊种布鲁氏菌043相比,羊种布鲁氏菌043-ΔBR0524缺失突变株的载菌量几乎没有下降(P0.05),对小鼠脾脏重量变化的影响也趋于相似(P0.05);侵染小鼠巨噬细胞试验结果与动物感染试验结果也趋于一致(P0.05),这表明BR0524基因的缺失不会对羊种布鲁氏菌043毒力功能的发挥产生显著的影响。

Construction and Preliminary Evaluation of Virulence of BR0524 Gene Deficient Brucella melitensis 043 Strain

To study the influence of BR0524 gene on the virulence of Brueella rnelitensis 043 strain,the BR0524 gene recombinant plasmid was constructed by replacing the gene BR0524 with kanamycin resistance gene based on the method of homologous recombination,and it was transformed into B.melitensis 043 competent cells.The BR0524 gene deficient Brucella melitensis 043 strain （043-Q78 BR0524）,which was identified by using the kanamycin resistance screening and detection primers. The 043-Q78BR0524 did not occur resilience mutation and genetic stability within 15 generations,then mice were infected with 043-Q78 BRO524,B.melitensis 043 and M5 strains （every mice was inoculated intraperitoneally （i.p.） with 106 CFU） and murine macrophage （RAW264.7）（MOI=100：l）.The results showed that the microbial load quantity of 043-Q78BR0524 did not decreased compare with the parental strain B.melitensis 043（P〈0.05）,and the influence on the spleen weight of mice was also tended to be similar, compared with the parental strain B.melitensis 043 （P〈0.05）.The results of the infection of mice macrophage experiments was also converge with animal infected test （P〈0.05）.These results indicated that the deficiency of BR0524 gene could not make a significant impact on the virulence of B.melitensis 043 strain.