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

发布时间：2018-01-05 14:29

  本文关键词：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　出处：《中华疾病控制杂志》2017年06期 　论文类型：期刊论文

  更多相关文章： 肠出血型大肠杆菌 抗体 单克隆 抗体特异性

【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%。
[Abstract]:Objective to express the fusion protein GST-Aat A of Escherichia coli O104: H4. Methods the transporter gene aat A of Escherichia coli O104: H4 was synthesized and ligated to p MDl8-T vector. E. coli DH5 伪 was transformed into E. coli DH5 伪, and the prokaryotic expression plasmid PGEX-6P-1-GST-Aat A was constructed. After sequencing, E. Coli BL21 was transformed into E. coli BL21. IPTG induced expression. SDS-PAGE and Western Blot were used to analyze the expression of the target protein and purify the recombinant protein. The purified fusion protein GST-Aat A was immunized with Balb/c mice. To prepare the corresponding monoclonal antibody; The purity, subtype and titer of the monoclonal antibody were determined. Results the prokaryotic expression vector PGEX-6P-1-GST-Aat A could express the fusion protein GST-Aat A. The purity of fusion protein GST-Aat A was 95% after purification by affinity chromatography. Conclusion A monoclonal antibody against the transporter Aat A was successfully prepared, which is a subtype of Ig G1 with good specificity and high titer. The purity can be as high as 97.
【作者单位】： 南方医科大学公共卫生学院三级生物安全实验室;
【基金】：广州市科技计划项目(2014Y2-00090)
【分类号】：R392-33
【正文快照】： (Chin J Dis Control Prev 2017,21(6):572-576)2011年5月15日,德国暴发肠出血型大肠埃希菌(Enterohemorrhagic escherichia coli,EHEC)O104∶H4的感染疫情,在本次疫情中,德国感染病例累计达4 047例,48人死亡[1-3]。EHEC O104∶H4致病力强[4],传播快且没有有效的治疗方法,引起，

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