鲍曼不动杆菌外膜蛋白A1S_0115的克隆、表达及免疫保护性研究  

Cloning, expression and antigen immune protection study of an outer membrane protein of severe infective Acinetobacter baumannii

作　　者：魏振波[1] 蔡昌芝[1] 杨赟 纪永军[1] 牟道华[1] 石云[1] 曾浩[1] 邹全明[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》2015年第5期380-385,共6页Immunological Journal

基　　金：国家科技重大专项子课题(2013ZX09J13107-03B)

摘　　要：目的构建鲍曼不动杆菌外膜蛋白A1S_0115胞外区(A1S_0115A)与GST标签融合表达的原核表达载体,在大肠杆菌XL-1 blue中表达、纯化A1S_0115A蛋白后,进行抗原免疫保护性的初步研究。方法利用生物信息学技术分析A1S_0115蛋白结构,确定蛋白的胞外区片段,PCR扩增该基因片段后,构建重组表达载体p GEX-6P-2-A1S_0115A,将重组载体转化大肠杆菌XL-1blue,IPTG诱导表达GST-A1S_0115A融合蛋白,采用GST亲和层析填料纯化并酶切获得A1S_0115A蛋白。利用已建立的Balb/c小鼠鲍曼不动杆菌全身感染模型对该蛋白抗原的免疫保护性进行初步评价。结果重组质粒经过Bam HⅠ和NotⅠ双酶切鉴定、核酸序列测定和IPTG诱导表达及酶切后蛋白的SDS-PAGE分析表明,A1S_0115A蛋白胞外区相对分子质量大小约50 000,GST标签相对分子质量大小约26 000,与预期设想符合,目的蛋白纯度在95%以上。用A1S_0115A蛋白对小鼠进行2轮次免疫保护性实验及其抗体的被动免疫实验,免疫攻毒后小鼠的保护率分别为50%和55%,被动免疫保护率为45%和50%。结论成功构建重组表达载体p GEX-6P-2-A1S_0115A,利用大肠杆菌可溶表达系统、GST亲和层析和酶切方法获得了高纯度的A1S_0115A蛋白。动物免疫攻毒保护实验结果表明A1S_0115A蛋白具有较好的免疫保护性,为研制新型有效的鲍曼不动杆菌疫苗奠定实验基础。This study aimed to construct an expression plasmid for the extracellular fragment of A1S_0115gene and identify the recombinant protein expression, and evaluate the preliminary immunoprotection of A1S_0115active fragment through animal trial. Firstly, bioinformatics was used to predict A1S_0115, and confirm the extracellular fragment of A1S_0115 protein. Then, the A1S_0115A＇s coding sequence was amplified by PCR and subcloned into p GEX-6p-2 vector. After the target gene was sequenced, the plasmid was transformed into E. coil XL-1 blue and induced with IPTG to express fusion protein A1S_0115A/GST, which subsequently purified by GST affinity chromatography method and digested by enzymes. SDS-PAGE analysis showed the expected molecular mass was about 50 000 and the purity of the expected protein reached to 95%. In two animal trials, the immunoprotection rates of A1S_0115A were 50% and 55%, while passive immunoprotection were 45% and 50%, respectively. Taken together, this study lays the experimental foundation for the development of the new and effective Acinetobacter baumannii vaccine.

关 键 词：鲍曼不动杆菌(Acinetobacter baumannii) A1S_0115A蛋白 GST融合蛋白 免疫保护性 

分 类 号：R392.1[医药卫生—免疫学]