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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。
Objective To prepare MDIi polyclonal antibody and identify its reactivity with tissue antigen MDIi. Methods The prokaryotic expression vector pET-32a / MDIi was constructed by PCR amplification and transformed into E. coli for expression. After identification of the expression product, the anti-MDIi polyclonal antibody was prepared from the immunized mice. The antibody titer was determined by indirect ELISA and identified by Western blot Antibody specificity, indirect ELISA detection of antibody and Cairina moschata MDIi response intensity. Results The prokaryotic expression vector pET-32a / MDIi was successfully constructed and the MDIi recombinant protein with a molecular weight of about 40 000 was expressed. The specific polyclonal antibody titer 1:128 000 was obtained The MDI reaction titers of antibodies, antibodies and Cairina moschata were 1:32 000. Conclusion High titer anti-MDIi polyclonal antibody was successfully prepared. The antibody and tissue antigens have strong immunoreactivity.