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目的:分析一个新的人突触相关蛋白(FRG4)抗原表位并制备其多克隆抗体。方法:从人胎肝文库PCR扩增获得FRG4基因全长cDNA序列;通过生物信息学分析,预测FRG4编码氨基酸序列的二级结构、抗原决定簇、功能结构域,并进行了多序列比对;采用固相多肽合成法合成了FRG4抗原多肽,并免疫家兔;用免疫组化检测蛋白在人肝癌细胞HepG2细胞中的表达。结果:通过生物信息学分析选取抗原13肽PKLVKEEVFWRNY,制备兔抗人FRG4多克隆抗体。高效液相色谱检测显示抗体纯度达82.79%,抗体滴度为1∶16000,Westernblot证实该抗体具有较好的反应性和特异性,免疫组化证实其主要在HepG2细胞胞浆中表达。结论:成功制备了新的人突触相关蛋白(FRG4)多克隆抗体。
Objective: To analyze a novel human synaptophysin (FRG4) epitope and prepare its polyclonal antibody. METHODS: The full-length cDNA sequence of FRG4 gene was obtained by PCR amplification from human fetal liver library. The secondary structure, antigenic determinant and functional domain of the amino acid sequence of FRG4 were predicted by bioinformatics analysis and multiple sequence alignment was performed. The FRG4 antigen peptide was synthesized by solid phase peptide synthesis method and immunized rabbits. The protein expression in HepG2 cells was detected by immunohistochemistry. Results: The bioinformatics analysis of the antigen peptide 13 PKLVKEEVFWRNY selected rabbit anti-human FRG4 polyclonal antibody. High performance liquid chromatography showed that the purity of the antibody reached 82.79%, the titer of the antibody was 1:16000, and Westernblot showed that the antibody had good reactivity and specificity. The results of immunohistochemistry showed that the antibody was mainly expressed in the cytoplasm of HepG2 cells. Conclusion: A new polyclonal antibody of human synaptophysin (FRG4) was successfully prepared.