论文部分内容阅读
目的克隆人syncytin基因并在大肠杆菌中表达,并用表达后的融合蛋白制备syncytin蛋白多克隆抗体。方法 PCR扩增人syncytin基因编码区的DNA片段,将其克隆入原核表达质粒pET30a(+),转化大肠杆菌BL21,诱导产生syncytin-His融合蛋白。采用割胶回收的方法纯化目的蛋白,免疫新西兰白兔,制备多克隆抗体。通过ELISA、Western-Blot和免疫组织化学等方法来检测抗体的灵敏度和特异性。结果成功表达并纯化了syncytin-His融合蛋白,SDS-PAGE分析表明融合蛋白主要以包涵体形式存在;ELISA法测定抗体效价为1:10 000;Western-Blot和免疫组织化学结果显示所制备的抗体能特异性识别syncytin蛋白。结论成功制备和鉴定了人syncytin多克隆抗体,多克隆抗体特异性强和效价高,为下一步研究Syncytin的生物学功能奠定了基础。
Objective To clone the human syncytin gene and express it in E. coli, and then use the expressed fusion protein to prepare syncytin protein polyclonal antibody. Methods The DNA fragment of human syncytin gene was amplified by PCR and cloned into prokaryotic expression plasmid pET30a (+). The recombinant plasmid was transformed into E. coli BL21 to induce syncytin-His fusion protein. The target protein was purified by tapping method and immunized New Zealand white rabbits to prepare polyclonal antibody. The sensitivity and specificity of the antibodies were tested by ELISA, Western-Blot and immunohistochemistry. Results The syncytin-His fusion protein was successfully expressed and purified. SDS-PAGE analysis showed that the fusion protein was mainly in the form of inclusion bodies. The antibody titer was 1:10 000 by ELISA. The results of Western-Blot and immunohistochemistry showed that the prepared fusion protein Antibodies specifically recognize the syncytin protein. Conclusion The human syncytin polyclonal antibody was successfully prepared and identified. The polyclonal antibody is highly specific and high titer, which lays the foundation for the further study on the biological function of Syncytin.