【摘要】 目的制备抗疣鼻栖鸭恒定链(MDIi)多克隆抗体,鉴定其与组织抗原MDIi的反应性。方法以PCR扩增获得MDIi序列构建原核表达载体pET-32a/MDIi,转化大肠杆菌进行表达;对表达产物鉴定后,免疫小鼠制备抗MDIi多克隆抗体;间接ELISA测定抗体效价,Western blot鉴定抗体特异性,间接ELISA检测抗体与疣鼻栖鸭组织MDIi的反应强度。结果成功构建了pET-32a/MDIi原核表达载体,诱导表达了相对分子质量(Mr)约40 000的包涵体形式的MDIi重组蛋白,免疫小鼠获得效价为1∶128 000的特异性多克隆抗体,抗体与疣鼻栖鸭组织中的MDIi反应滴度为1∶32 000。结论成功制备了高效价的特异性抗MDIi多克隆抗体,抗体与组织抗原有较强的免疫反应性。更多还原

【Abstract】 Objective To prepare polyclonal antibody against invariant chain of Muscovy duck(Cairina moschata)(MDIi) and identify its reaction with MDIi extracted from tissues of Muscovy duck. Methods MDIi was amplified by PCR and used to construct the prokaryotic expression vector of pET-32a/MDIi by linking with the plasmid of pET-32a. Then pET-32a/MDIi was transformed into E. coli Rosetta to induce the prokaryotic expression. After identified by SDS-PAGE,prokaryotic expression products were further purified from running gel of SDS-PAGE and injected into mice to prepare polyclonal antibody against MDIi. The titer and specificity of the polyclonal antibody against MDIi were analyzed by indirect ELISA and Western blotting,respectively. The intensity of reaction between the polyclonal antibody and MDIi extracted from tissues of Muscovy duck was also identified by indirect ELISA. Results The prokaryotic expression vector pET-32a/MDIi was successfully constructed. About 40 kD recombinant proteins of MDIi were confirmed to be expressed in the form of inclusion body in Rosetta. Polyclonal antibody against MDIi with a titer of 1∶128 000 was obtained from the immunized mice and its high specificity was demonstrated by Western blotting. The titer of reaction between the polyclonal antibody and MDIi was1∶32 000. Conclusion The polyclonal antibody against MDIi was successfully prepared with a high titer and specificity. It has a strong immune reaction with MDIi extracted from tissues of Muscovy duck.更多还原