GPC-3蛋白N端可溶性片段多克隆抗体的制备及鉴定

Prokaryotic expression of soluble NH2-terminal fragment of glypican-3 and preparation of anti-GPC3 antibody

目的:通过原核表达并纯化GPC3-N端蛋白及根据分析GPC3-N端蛋白可能的抗原表位合成多肽,免疫家兔获得抗-GPC3多克隆抗体,为深入研究GPC3基因的功能及其临床应用奠定基础。方法:将GPC3-N端基因片段亚克隆至pQE30原核表达载体,表达带有6-His的GPC3的融合蛋白质,纯化蛋白后免疫家兔;同时应用Goldkey软件预测分析GPC3蛋白N端可能的抗原表位,根据分析结果合成多肽命名为lw1,免疫家兔,获得抗-GPC3多克隆抗体,并鉴定其特性。结果:成功表达出GPC3-N端蛋白,用纯化后的蛋白及合成的肽免疫家兔,获得特异性兔抗多克隆抗体,通过间接ELISA,Western blot和免疫荧光技术证实该多抗能识别原核表达的外源性及HepG2细胞内源性GPC3蛋白。结论:通过两种方法获得的蛋白免疫家兔都能获得能特异识别GPC3蛋白的多克隆抗体,将为GPC3基因的功能研究提供研究工具,并为临床原发性肝癌的血清学早期诊断提供新的标记物。

Objective：To obtain NH2-terminal protein of GPC3 and prepare the anti-GPC3 polyclonal antibody,and provide theoretical basis for further study and clinical utilization of GPC3 gene.Methods：Recombinant plasmid pQE30-GPC3-N was constructed by inserting the GPC3 coding sequences into vector pQE30,and GPC3 protein was successfully expressed with 6-histidines tag.With Goldkey Softwar,we can Forecast and Analysis e GPC3 N protein epitope possible,according to analysis of synthetic peptide named lw1,immune rabbitthe purified protein was used as a immunogen to the rabbit,and then GPC3 polyclonal antibody was identified by Western blot,elisa and immunofluorescent microscopy.Results：GPC3-N protein was expressed in prokaryotic cells;the polyclonal antibody was obtained and identified from immunize rabbit;the antibody could specially recognized the ectogenetic and endogenetic GPC3 protein in prokaryotic and eukaryotic cells（such as HepG2）.Conclusion：The successful expression of GPC3-N fusion protein and preparation of the polyclonal antibody will provide material for further studies of GPC3 gene function and GPC3 is a novel serological marker essential for the early detection of HCC.