小鼠多囊蛋白1氨基端多克隆抗体的制备和其生物特性分析

Preparation and characterization of a polyclonal antibody against mouse NH2terminal domain of polycystin-1

目的制备针对多囊蛋白1(PC1)胞外区的抗体,为分析PC1胞外区生物特性和功能提供工具。方法根据生物信息学分析结果,PCR扩增编码小鼠Pkd1的氨基端474E-640L的cDNA片段。将该片段克隆到GST融合蛋白表达载体上,在IPTG诱导下产生小鼠Pkd1抗原(mPkd1-N)。纯化浓缩目的蛋白并制备兔抗PC1氨基端多克隆抗体(mPkd1-Np),并以Westernblot法、免疫组化以及免疫荧光方法分析该兔抗PC1氨基端抗体的特异性。结果成功地构建了mPkd1-N片段真核及原核表达载体,在大肠杆菌中实现表达,并制备了抗小鼠PC1氨基端的多克隆抗体mPkd1-Np。通过生化和细胞学方法证实了该抗体针对PC1的特异性。结论成功制备了高效价、特异性的兔抗mPkd1-Np多克隆抗体。

Objective To produce a rabbit polyclonal antibody, mPkdl-Np, against the extracellular portions of polycys- tin-] （PC]） in order to explore the functional roles of the PC1 NH2-terminus. Methods Based on hydrophobic/hydrophilic analyses, we chose a cDNA fragment that encodes amino acids 474E-O40L on PC1 and amplified it via RT-PCR. The PCR product was then cloned into a prokaryotic expression vector pGEX-GST. After IPTG induction, the antigen mPkd]-N was produced and further purified. A rabbit was immunized with this antigen and its antiserum was collected. The mPkd]-Np anti- body was validated to be specific for PC] protein through Western blotting, immunohistochemistry, and immunofluorescence methods. Results The prokaryotic expression vector pGEX-mPkdi-N was successfully constructed and mPkdl-N antigen was induced to express in E. coil Rossetta cells. Using this antigen, the polyclonal antibody mPkdl-Np was produced and its specificity for PC1 was proved through biochemistry and cellular assays. Conclusion We successfully produced an anti-PC1 NH2-terminal polyclonal antibody named mPkdl-Np. The polyclonal antibody provides a platform for further research into PC1 NH2-terminal function, specifically renal tubulogenesis and its maintenance.