木薯14-3-3蛋白家族成员MeGRF3基因的原核表达及多克隆抗体制备

14-3-3蛋白是植物体内重要的信号转导调节分子,在碳代谢、逆境胁迫响应、生长发育等过程中发挥重要调控作用。为了深入解析14-3-3蛋白家族在木薯中的生物学功能,该研究采用酶切连接方法构建木薯14-3-3蛋白家族成员MeGRF3的原核表达载体,用热激法转化大肠杆菌Rosetta(DE3)株系,诱导表达带有MeGRF3的融合蛋白;使用纯化后的MeGRF3融合蛋白免疫新西兰公兔制备多克隆抗体,并检测抗体的效价和特异性。结果显示:(1)成功构建了木薯MeGRF3的原核表达载体pET-30a-MeGRF3,并诱导表达得到带有MeGRF3和6个His标签的融合蛋白。(2)MeGRF3融合蛋白主要以包涵体的形式存在,相对分子质量约为33 kD。(3)间接酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)测定的抗体效价为1024000。(4)Western blot分析显示,木薯叶片、茎尖、茎皮和块根中均检测到与MeGRF3蛋白大小一致的条带,说明MeGRF3在这4个组织器官中均表达,但主要是在茎尖和块根中大量积累,表明该研究成功制备的MeGRF3多克隆抗体的特异性较好。

Prokaryotic Expression and Polyclonal Antibody Preparation of Cassava 14 3 3 Protein Family Member MeGRF3 Gene

14 3 3 protein is an important signal transduction regulator in plants and plays significant roles in carbon metabolism, stress response, growth and development, etc. In order to in depth analysis the biological function of 14 3 3 protein family in cassava, we constructed the prokaryotic expression vector pET 30a MeGRF3 of MeGRF3, a member of cassava 14 3 3 protein family, via enzymes digestion and ligation, and then transformed it into Escherichia coli Rosetta (DE3) strain by heat shock method to induce the expression of MeGRF3 fusion protein. The purified MeGRF3 fusion protein was used to immunize the New Zealand male rabbits to prepare the polyclonal antibody, and then the titer and specificity of polyclonal antibody were detected. The results showed that: (1) the prokaryotic expression vector pET 30a MeGRF3 of cassava MeGRF3 was successfully constructed, and the fusion protein with MeGRF3 and six His tags was induced and expressed. (2) The MeGRF3 fusion protein was mainly in the form of inclusion body, and its molecular weight was about 33 kDa. (3) The antibody titer determined by indirect enzyme linked immunosorbent assay (ELISA) was 1 024 000. (4) The bands with the same molecular weight of MeGRF3 were detected in leaves, stems, barks and roots of cassava via Western blot, indicating that MeGRF3 was expressed in all of the four tissues, but mainly accumulated in the stems and roots. The results showed that the MeGRF3 polyclonal antibody successfully prepared in this study had good specificity.