Preparation and identification of monoclonal antibody against EHEC O104: H4 AatA transporter protein
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摘要: 目的 表达肠出血型大肠埃希菌O104 ：H4的融合蛋白GST-AatA，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aatA，连接至pMDl8-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-AatA，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-AatA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-AatA能表达融合蛋白GST-AatA；亲和层析纯化后融合蛋白GST-AatA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AatA的单克隆抗体，该抗体为IgG1亚型，特异性好，效价高，纯度可达97%。Abstract: Objective To express EHEC O104 :H4 GST-AatA fusion protein and to obtain the monoclonal antibody specific for AatA. Methods The aatA gene sequence was synthesized and connected to pMD18-T vector, followed by the transformation of E.coli DH5α. 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.coli 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 monoclonal antibody was obtained from the immunized Balb/c mouse. Conclusions A specific and high-titer monoclonal antibody was produced with a purity of 97% after purification.
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