Chen Yunyu, Niu Xiayi, Lin Yuan, Liu Xiaoping
Center for New Drug Screening & Evaluation, School of Pharmacy, Wannan Medical College, Wuhu 241002; Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China.
Center for New Drug Screening & Evaluation, School of Pharmacy, Wannan Medical College, Wuhu 241002, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2018 Oct;34(10):942-948.
Objective To prepare recombinant Escherichia coli filamentous thermosensitive protein Z-interacting protein A (Ec-ZipA) 185-328 amino acid functional protein and its specific rat polyclonal antibody. Methods The synthetic Ec-ZipA gene fragment was successfully cloned into pET-30a(+) vector. After transformation, recombinant Ec-ZipA was stably expressed under an optimal bacterial expression condition in E.coli BL21(DE3) cells, and purified by HisTrap affinity chromatography. Then the biological activity of purified Ec-ZipA was further analyzed by fluorescence polarization (FP). The anti-Ec-ZipA polyclonal antibody was prepared using recombinant Ec-ZipA as antigen to vaccinate rat subcutaneously. The titer and immunological specificity of the polyclonal antibody were determined by ELISA and Western blot analysis. Results The results of SDS-PAGE and Western blot showed that recombinant Ec-ZipA was successfully expressed and purified. The FP demonstrated a perfect biological activity for purified Ec-ZipA. The titer of polyclonal antibody was 1:512000 by ELISA, and Western blot confirmed high sensitivity and specificity of polyclonal antibody against recombinant and endogenous Ec-ZipA respectively. Conclusion The prokaryotic expression, and purification of Ec-ZipA is performed, and rat anti-Ec-ZipA polyclonal antibody is successfully prepared.
目的 制备重组大肠杆菌丝状热敏蛋白Z相互作用蛋白A（Ec-ZipA）185-328氨基酸功能蛋白及其特异性大鼠多克隆抗体。方法 将合成的Ec-ZipA基因片段成功克隆到pET-30a(+)载体中。转化后，重组Ec-ZipA在最佳细菌表达条件下于大肠杆菌BL21(DE3)细胞中稳定表达，并通过HisTrap亲和层析进行纯化。然后通过荧光偏振（FP）进一步分析纯化后的Ec-ZipA的生物学活性。以重组Ec-ZipA为抗原皮下免疫大鼠制备抗Ec-ZipA多克隆抗体。通过ELISA和Western印迹分析测定多克隆抗体的效价和免疫特异性。结果 SDS-PAGE和Western印迹结果表明重组Ec-ZipA成功表达并纯化。FP显示纯化后的Ec-ZipA具有良好的生物学活性。ELISA检测多克隆抗体效价为1:512000，Western印迹证实多克隆抗体分别对重组和内源性Ec-ZipA具有高敏感性和特异性。结论 实现了Ec-ZipA的原核表达和纯化，并成功制备了大鼠抗Ec-ZipA多克隆抗体。
