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目的制备鲍曼不动杆菌(Ab)A1S_1969重组蛋白,利用小鼠全身脓毒血症感染致死模型评价A1S_1969重组蛋白的免疫保护效果,探讨可能的免疫保护机制,为筛选Ab疫苗有效的保护性抗原奠定基础。方法基于反向疫苗学技术筛选出Ab外膜蛋白A1S_1969,利用p GEX-6P-2质粒构建GST融合表达载体,重组表达的A1S_1969蛋白经亲和层析高效纯化后与铝佐剂吸附制备而成重组A1S_1969免疫原;采用Balb/c小鼠全身感染模型评价A1S_1969重组蛋白的免疫保护效果,通过ELISA检测免疫小鼠Ig G抗体滴度和Ig G抗体亚型。制备A1S_1969重组蛋白抗血清进行体外调理吞噬杀菌实验。结果成功克隆、表达并纯化A1S_1969重组蛋白,纯度>90%。动物免疫保护结果显示A1S_1969重组蛋白组小鼠存活率为55.6%,对照组小鼠存活率为20.0%(P<0.05);末次免疫7 d后小鼠体内总Ig G抗体滴度为1∶64 000,以Ig G1亚型为主;体外调理吞噬杀菌实验结果显示A1S_1969特异性血清抗体可以增强中性粒细胞对Ab的杀菌作用,实验组杀菌率可达55%,对照组无杀菌活性。结论 A1S_1969重组蛋白可显著提高全身脓毒血症感染致死模型小鼠的存活率,具有良好的免疫保护效果。
Objective To prepare the recombinant protein A1S1969 of Acinetobacter baumannii (AB) and evaluate the immunoprotective effect of A1S_1969 recombinant protein on the lethal model of systemic sepsis in mice. To explore the possible mechanism of immune protection, Lay the foundation. Methods The Ab outer membrane protein A1S_1969 was screened based on the reverse vaccine technology. The GST fusion expression vector was constructed by using pGEX-6P-2 plasmid. The recombinant protein A1S_1969 was purified by affinity chromatography and adsorbed onto the aluminum adjuvant Recombinant A1S_1969 immunogen was used to evaluate the immunoprotective effect of A1S_1969 recombinant protein in Balb / c mouse model of systemic infection. Ig G antibody titer and Ig G antibody subtype of immunized mice were detected by ELISA. Preparation A1S_1969 recombinant protein antisera for in vitro conditioning phagocytosis experiments. Results The recombinant protein A1S_1969 was successfully cloned, expressed and purified with purity> 90%. The results of animal immunization showed that the survival rate of mice in the A1S_1969 recombinant protein group was 55.6%, and that of the control group was 20.0% (P <0.05); the total IgG antibody titer in mice after the last immunization was 1:64 000, Ig G1 subtype mainly; in vitro opsonization phagocytosis test results show that A1S_1969 specific serum antibody can enhance neutrophil bactericidal effect on Ab, the experimental group sterilization rate of up to 55%, the control group without bactericidal activity. Conclusion A1S_1969 recombinant protein can significantly increase the survival rate of mice with lethal sepsis-induced death and has good immunoprotective effect.