布鲁氏菌M5-90 bp26表位缺失株的构建及对肝脏的影响研究

Construction and Studies on the Effect upon Liver of Brucella Epitope-deleted Strain M5-90 bp26

目的:构建羊种布鲁氏菌表位缺失株M5-90 bp26,以小鼠的肝脏为模型进行毒力评价。方法:利用SacB负筛选标记的方法构建两株M5-90 bp26表位缺失株并进行生物学性状和Western Blot验证;用3.0×106CFU/0.2mL M5-90疫苗株及缺失株分别免疫BALB/c小鼠,在10d、20d、30d处死小鼠,HE染色观察肝脏的病理变化。结果:经测序和Western Blot分析成功获得M5-90Δbp261、M5-90Δbp262两株突变株;常规生物学鉴定未发生生物学性状的改变;HE染色表明bp26缺失株毒力均低于疫苗株M5-90。结论:结合CFU计数及脾脏指数首次证实肝脏可作为毒力判定的标准;构建的布鲁氏菌基因突变株M5-90Δbp262具有毒力弱,并具有血清学鉴别诊断的"标签"作用,可作为未来鉴别诊断新型布鲁氏菌疫苗的候选株。

Objective ：To construct Brucella epitope- deleted strain M5 -90 bp26 and evaluate the virulence of mutant strains using mice liver as model. Method： The two epitope - deleted strains M5 - 90 bp26 were constructed using SacB as negative selectable marker gene and verificated by Biological characteristics andWestern Blot. The BALB/c mice were immunized with Brucella vaccine strain M5 -90 and mutant strains at the doses of 3.0 × 10s CFU/0. 2mL, then the mice were killed after they were immuned after 10d ,20d and 30d. Pathologic changes of liver were detected by HE staining. Result：The two mutant strains M5 -90Abp261 and M5 -90Abp262 were successfully ob- tained by sequencing and Western Blot. Biological characteristics didn＇ t change of mutants compared to parant strain with conventional bacteriological method. The results of HE staining showed that the virulence of bp26 mutant strains were lower than that of vaccine strain M5 -90. Conclusion：Compared with CFU Counting, and spleen index pathological change of liver could be firstly employed to candidate standard of virulence. B. melitensis M5 - 90Abp262 in this study had the characteristic of lower virulence and had the ＂tag＂ effect of Sero- logical diagnosis,which might be used as candidate strain for diagnosis of new vaccine in the future.