鲍曼不动杆菌外膜蛋白A1S1969的克隆表达及免疫保护机制初步研究  

The cloning,expression and possible immune protection mechanism of Acinetobacter baumannii A1S_1969 outer membrane protein

作　　者：蔡昌芝[1] 冯强[1] 杨赟 魏振波[1] 纪永军[1] 牟道华[1] 敬海明[1] 邹全明[1] 曾浩[1] 

机构地区：[1]第三军医大学药学系微生物与生化药学教研室暨国家免疫生物制品工程技术研究中心,重庆400038

出　　处：《免疫学杂志》2016年第2期104-108,共5页Immunological Journal

基　　金：国家重大科技专项(2013ZX09J13107-03B)

摘　　要：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。In this study, we aimed to evaluate the immune protection of recombinant protein AIS_1969 from Acinetobacter baumannii on a systemic Acinetobacter infection mouse model. Outer membrane protein A1S_1969 gene from GenBank was cloned in pGEX-6p-2 plasmids, expressed in E.coli and purified by GST-affinity chromatography. Balb/c mice were immunized with recombinant A1S_1969 protein, and then challenged with Ab-17978 in 4.2× 108 CFU/ml through intraperitoneal injection. The mouse survival rate of A1S_1969 immunized group （56.6%） was significant higher than that of the control group （20%） （P〈0.05）. In addition, A1S_1969 recombinant protein immunization elicited a highly-titered IgG （1 ： 64 000） responses, mainly in IgG1 subclass, and the specific IgG antibodies enhanced bacterial elimination in mouse bloods. Taken together, A1S_1969 recombinant protein may protect mouse from lethal Ab infection and be worthy of further investigation as a candidate for Ab vaccine.

关 键 词：鲍曼不动杆菌 基因工程疫苗 克隆表达 重组蛋白 免疫保护 

分 类 号：R392-33[医药卫生—免疫学]