布鲁氏菌部分毒力相关调控因子免疫原性检测与保护性评价

Detection of cellular immunogenicity to the virulence-associated transcriptional regulators of Brucella

目的检测部分布鲁氏菌毒力相关调控因子刺激机体产生细胞免疫反应的能力,并对具有细胞免疫原性蛋白的免疫保护性进行初步评价。方法利用Gateway的方法将9个布鲁氏菌毒力相关转录调控因子克隆至表达载体pHXGWA上并利用Ni-NAT层析的方法纯化重组蛋白。构建布鲁氏菌疫苗株S19株免疫的BALB/c小鼠模型,免疫35d后分离小鼠脾细胞并用体外重组蛋白再刺激,ELISA方法检测蛋白体再刺激后小鼠脾细胞分泌的细胞因子IFN-γ水平,了解毒力相关调控因子刺激机体产生细胞免疫反应情况。以CpG-ODN为免疫佐剂,用具有细胞免疫活性蛋白免疫小鼠,观察对布鲁氏菌强毒株544攻击感染的免疫保护作用。结果 7个布鲁氏菌毒力相关调控因子在大肠埃希菌中得以表达。其中BMEⅠ1573及BMEⅡ0475再刺激下S19免疫小鼠脾细胞产生的IFN-γ水平高于PBS对照小鼠。而在以CpG-ODN为免疫佐剂时,两者均不具有免疫保护性。结论布鲁氏菌LysR家族及GntR家族的转录因子BMEⅠ1573及BMEⅡ0475具有细胞免疫原性,初步检测无免疫保护作用。

Objectives To detect cellular immunoreaction to the virulence-associated transcriptional regulators of Brucella and evaluate the immunoprotection provided by cells with immunogenic proteins. Methods Nine transcriptional regu lators of Brucella were cloned into the expression vector pHXGWA using the Gateway system and the recombinant proteins were purified using Ni-NAT. An animal model was established by immunizing BAL B/c mice with the live vaccine strain S19 of Brucella. The ability of the proteins to induce the production of gamma interferon in mice was detected with ELISA using protein re-stimulation in vitro. The immunoprotection provided by cells with immunogenic proteins was pre- liminarily evaluated using a mouse, model of a brucella 544 challenge. Results Seven transcriptional regulators were successfully expressed in E. coli and purified. The proteins BME Ⅰ 1573 and BME Ⅱ0475 induced a stronger IFN-γ response in S19 immunized mice than in PBS control mice. Unfortunately, none of the proteins provided protection against a chal- lenge with Brucella 544 in which CpG-ODN served as the adjuvant. Conclusion The GntR and LysR transcriptional regulators BME Ⅱ 0475 and BME Ⅰ 1573 provide cellular immunogenicity but do not provide protection against Brucella.