肠出血型大肠埃希菌O104∶H4转运蛋白AatA单克隆抗体制备及鉴定  

Preparation and identification of monoclonal antibody against EHEC O104:H4 Aat A transporter protein

作　　者：邵娜[1] 李以圣 王湘雨[1] 华颖[1] 万成松[1] 

机构地区：[1]南方医科大学公共卫生学院三级生物安全实验室,广东广州510515

出　　处：《中华疾病控制杂志》2017年第6期572-576,共5页Chinese Journal of Disease Control & Prevention

基　　金：广州市科技计划项目(2014Y2-00090)

摘　　要：目的表达肠出血型大肠埃希菌O104∶H4的融合蛋白GST-Aat A,获得单克隆抗体。方法人工合成肠出血型大肠埃希菌O104∶H4的转运蛋白基因aat A,连接至p MDl8-T载体,转化E.coli DH5α,双酶切回收,构建原核表达质粒PGEX-6P-1-GST-Aat A,测序鉴定后,转化E.Coli BL21,IPTG诱导表达,SDS-PAGE和Western Blot分析目的蛋白的表达并纯化该重组蛋白。将纯化后的融合蛋白GST-Aat A免疫Balb/c小鼠,制备相应单克隆抗体;并对该单克隆抗体的纯度、亚型、效价、特异性进行鉴定和分析。结果构建的原核表达载体PGEX-6P-1-GST-Aat A能表达融合蛋白GST-Aat A;亲和层析纯化后融合蛋白GST-Aat A纯度达到95%,免疫小鼠后得到相应特异性单克隆抗体。结论成功制备出一株抗转运蛋白Aat A的单克隆抗体,该抗体为Ig G1亚型,特异性好,效价高,纯度可达97%。Objective To express EHEC O104 ： H4 GST-AatA fusion protein and to obtain the monoclonal anti- body specific for AatA. Methods The aatA gene sequence was synthesized and connected to pMD18-T vector, followed by the transformation of E. coli DHSa. The recycled PCR product was inserted into the prokaryotic plasmid PGEX-6P-1 and confirmed by DNA Sequencing. The constructed prokaryotic expression vector PGEX-6P-1-GST-AatA was used to transform E. eoli BL21. IPTG was added to induce the expression of fusion protein. The purified protein was analyzed by SDS-PAGE and Western Blot. The monoclonal antibody was obtained by immunizing Balb/c mouse with purified GST-AatA protein and was examined for the purity, subtypes, specificity and titer thereafter. Results The prokaryotic expression vector PGEX- 6P-1-GST-AatA was successfully constructed. The purity of the fusion protein was about 95%. The specific monoelonal antibody was obtained from the immunized Balb/e mouse. Conclusions A specific and high-titer monoclonal antibody was produced with a purity of 97% after purification.

关 键 词：肠出血型大肠杆菌 抗体 单克隆 抗体特异性 

分 类 号：R117[医药卫生—公共卫生与预防医学]