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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。
Objective To detect the ability of part of brucellosis-related regulatory factors to stimulate cellular immune response and to evaluate the immunoprotective properties of cellular immunogenic proteins. Methods Nine strains of Brucella virulence-associated transcriptional regulators were cloned into the expression vector pHXGWA using Gateway method and the recombinant proteins were purified by Ni-NAT chromatography. The BALB / c mouse model immunized with the Brucella vaccine strain S19 was constructed. After 35 days of immunization, the splenocytes of mice were isolated and re-stimulated with recombinant protein in vitro. The levels of cytokines IFN secreted by mouse spleen cells -γ level to understand the virulence-related regulatory factors to stimulate the body to produce cellular immune response. CpG-ODN was used as an adjuvant to immunize mice with cellular immunocompetent proteins to observe the protective immunity against Brucella virulent 544 challenge infection. Results Seven Brucella virulence-associated regulatory factors were expressed in Escherichia coli. The spleen cells of S19-immunized mice stimulated by BMEⅠ1573 and BMEⅡ0475 were higher than that of PBS control mice. In the case of CpG-ODN as an adjuvant, neither was immunoprotective. Conclusion The Brucella LysR family and GntR family transcription factor BME Ⅰ 1573 and BME Ⅱ 0475 have cellular immunogenicity, and no initial immunoprotection was detected.