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目的:制备FPC-L蛋白的多克隆抗体,对PKHDL1基因产物进行初步的细胞生物学研究。方法:根据生物信息学分析结果,PCR扩增编码人类FPC-L的N端633L-768K的cDNA片段。将该片段克隆到GST融合蛋白表达载体上,在IPTG诱导下产生人类FPC-L抗原(hFL-N)。纯化目的蛋白并制备兔抗FPC-L蛋白多克隆抗体。Western blot鉴定抗体特异性,并以间接免疫荧光法初步分析该蛋白在细胞内定位。结果:成功地构建了hFL-N片段原核表达载体,在大肠杆菌中实现表达,并制备了hFPC-L蛋白的多克隆抗体hFL-Np。通过生化和细胞学方法证实了该抗体针对FPC-L的特异性和揭示了该蛋白的亚细胞定位。结论:成功制备了高效价并具特异性的兔抗FPC-L蛋白多克隆抗体,并揭示了该蛋白主要表达于细胞质内,为进一步研究PKHDL1基因产物的生物功能奠定了基础。
OBJECTIVE: To prepare polyclonal antibodies against FPC-L protein and to perform preliminary cell biology studies on PKHDL1 gene products. Methods: According to the results of bioinformatics analysis, a cDNA fragment encoding the N-terminal 633L-768K of human FPC-L was amplified by PCR. The fragment was cloned into the GST fusion protein expression vector to generate human FPC-L antigen (hFL-N) induced by IPTG. The target protein was purified and a polyclonal antibody to rabbit anti-FPC-L protein was prepared. The specificity of the antibody was identified by Western blot and the protein was localized intracellularly by indirect immunofluorescence assay. Results: The prokaryotic expression vector of hFL-N fragment was successfully constructed and expressed in E. coli. The hFL-Np polyclonal antibody of hFPC-L protein was also prepared. The specificity of the antibody against FPC-L was confirmed by biochemical and cytological methods and the subcellular localization of the protein was revealed. CONCLUSION: Polyclonal antibody against rabbit polyclonal antibody against FPC-L with high titer and specificity has been successfully prepared and revealed that the protein is mainly expressed in the cytoplasm, which lays the foundation for further study on the biological function of PKHDL1 gene product.