Liu Shengjie, Zhu Maoying, Chen Deyu, Wu Chao, Ni Qingsheng, Yu Weiyi
School of Life Sciences, Fuyang Normal College, Fuyang 236037; Anhui Key Laboratory of Zoonoses, Anhui Agricultural University, Hefei 230036, China.
School of Life Sciences, Fuyang Normal College, Fuyang 236037, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2014 Jun;30(6):623-6.
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.
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.
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 was 1:32 000.
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.
制备针对番鸭（Cairina moschata）恒定链（MDIi）的多克隆抗体，并鉴定其与从番鸭组织中提取的MDIi的反应。
通过PCR扩增MDIi，并将其与pET-32a质粒连接构建pET-32a/MDIi原核表达载体。然后将pET-32a/MDIi转化到大肠杆菌Rosetta中诱导原核表达。经SDS-PAGE鉴定后，从SDS-PAGE的运行胶中进一步纯化原核表达产物，并注射到小鼠体内制备抗MDIi的多克隆抗体。分别通过间接ELISA和Western印迹分析抗MDIi多克隆抗体的效价和特异性。还通过间接ELISA鉴定多克隆抗体与从番鸭组织中提取的MDIi之间的反应强度。
成功构建了原核表达载体pET-32a/MDIi。证实约40 kD的MDIi重组蛋白在Rosetta中以包涵体形式表达。从免疫小鼠中获得了效价为1:128 000的抗MDIi多克隆抗体，Western印迹证明其具有高特异性。多克隆抗体与MDIi的反应效价为1:32 000。
成功制备了效价高、特异性强的抗MDIi多克隆抗体。它与从番鸭组织中提取的MDIi有强烈的免疫反应。
