抗对硫磷基因工程新型抗体的制备及初步鉴定

Construction and preliminary identification of genetically engineered tetravalent antibodies against parathion

摘要:

摘要: 【目的】提高抗对硫磷抗体的亲和力以提高酶联免疫检测的灵敏度。【方法】本研究通过抗对硫磷单链抗体基因和核心链霉亲和素基因片段的拼接重组，获得抗对硫磷单链抗体-核心链霉亲和素融合基因（scfv-sa），并将该融合基因（scfv-sa）插入到表达载体pET28a(+)中，转化大肠杆菌BL21（DE3）进行原核表达，制备融合蛋白。SDS-PAGE和Western blot鉴定scfv-sa的表达，Ni+-NTA亲和层析柱纯化融合蛋白，并用ELISA方法测定该融合抗体的亲和力。【结果】结果表明，在大肠杆菌BL21（DE3）中该融合基因能表达出分子量约为46kD的融合蛋白，形成了四价结构域-四价聚合抗体。ELISA测定结果表明该抗体能与对硫磷特异结合，抗体效价在1:1×106以上，亲和常数为4.25×107 L /mol。 【结论】制备的抗对硫磷四价聚合抗体能与抗原特异结合，与单克隆抗体相比，抗原结合位点显著增加，ELISA检测灵敏度显著提高。

关键词:关键词: 对硫磷;链霉亲和素;四价抗体;原核表达

Abstract:

Abstract: [Objective]The objective is to improve the affinity of antibody against parathion with an aim to enhance the sensitivity of ELISA assay. [Methods] We recombined anti-parathion single-chain variable fragment gene (scfv) and the core streptavidin gene(sa) to produce an anti-parathion single-chain variable fragment-core of streptavidin-biotin fusion gene (scfv-sa), and inserted the fusion gene (scfv-sa) into the vector pET28a (+) in E.coli BL21 for transformation by prokaryotic expression. The expression was analyzed with SDS-PAGE and Western blot. the affinity of the recombinant protein was measured by ELISA after purified by metal affinity chromatography using Ni-NTA. [Results] The recombinant gene could express a fusion protein about 46 kDa, which formed a tetravalent domain, tetravalent antibody. ELISA results showed that the tetravalent antibody could bound to parathion specifically with the Affinity constant of 4.25 × 107 L / mol and titer of over 1:1×106. [Conclusion] The recombining anti-parathion tetravalent antibody could react with parathion specifically with significantly more binding sites with the monoclonal antibody, based on which the detection sensitivity of ELISA would be improved.

Key words:Keywords: parathion;core streptavidin ;tetravalent antibodies; prokaryotic expression