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【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,获得了抗对硫磷单链抗体-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46kDa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4.25×107L/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。
【Objective】 To improve the affinity of anti-parathion antibody to improve the sensitivity of enzyme-linked immunoassay. 【Method】 In this study, the scfv-sa gene encoding anti-parathion single chain antibody-core streptavidin was obtained by splicing and recombination of anti-parathion-single chain antibody gene and core streptavidin gene. , And the fusion gene (scfv-sa) was inserted into the expression vector pET28a (+) and transformed into E. coli BL21 (DE3) for prokaryotic expression to prepare a fusion protein. The expression of scfv-sa was identified by SDS-PAGE and Western blot. The fusion protein was purified by Ni + -NTA affinity chromatography and the affinity of the fusion protein was determined by ELISA. 【Result】 The results showed that the fusion gene expressed in E. coli BL21 (DE3) with a molecular weight of about 46 kDa and formed a tetravalent domain-tetravalent polyclonal antibody. The results of ELISA showed that the antibody could specifically bind to parathion. The antibody titer was above 1: 1 × 106 and the affinity constant was 4.25 × 107 L / mol. 【Conclusion】 The prepared tetravalence-para-parathion-polyvalent antibody can specifically bind to antigen. Compared with the monoclonal antibody, the antigen-binding site is significantly increased, and the sensitivity of ELISA detection is significantly increased.