高尔基体蛋白73原核表达系统的构建及单克隆抗体的制备.docxVIP

·301·．技术方法．高尔基体蛋1兰173原核表达系统的构建 ·301· ．技术方法． 高尔基体蛋1兰173原核表达系统的构建 及单克隆抗体的制备 郭静霞，魏红山，宋永继，赵静，刘爱霞，刘佳，陈霖，徐军，李伯安，毛远丽 【摘要】 目的克隆、表达高尔基体蛋白73(GP73)，并制备单克隆抗体。方法用逆转录聚合 酶链反应法从人肝癌细胞系HepG2细胞中扩增GP73编码序列，将产物与pQE31载体连接，转化入 大肠埃希菌B121，诱导表达后通过His标签进行纯化，用纯化后的蛋白免疫BALB／c小鼠，采用常规 的细胞融合技术制备单克隆抗体，并对其进行鉴定。结果重组载体经序列分析与报道序列同源性 100％，并获得了重组蛋白。筛选出5株能稳定分泌抗GP73的单克隆抗体杂交瘤细胞株，免疫球蛋白 类型有2株为IgGl类。Western Blot印迹显示这些单抗能特异结合GP73蛋白。结论成功构建了 人高尔基体蛋白73原核表达体系，并获得了单克隆抗体。 【主题词】 高尔基体；蛋白质73；克隆，分子；抗体，单克隆；肝肿瘤 Cloning and expressing of Goigi protein73 gene fragment and preparation of monoclonal antibodies against the recombinant protein GUO Jing—xia。，WEI Hong—shan，SONG y0增-ji，ZHAO Jing，LIU Ai- xia，LIU Jia，CHEN Lin，XU Jun，LI Bo—an，MAO Yuan—li．‘Ce，咖r of Clinical Laboratory，The No．302 Hospital of The PLA，Beqing 100039，China Corresponding author：LI Bo-口n，Email：lba@263．net；MAO Yuan—li，Email：maoyuanlee@yahoo．corn 【Abstract】0b|eetive To clone and express human Golgi glycoprotein73 protein，and prepare the monoclonal antibody(mAb)against the protein．Methods GP73 gene was amplified from HepG2 cells by RT-PCR，then ligated with pQE3 1 form recombinant plasmid pQE-GP73 and transformed into E．coli BL21．The protein induced by IPTG was purified by 6×His·tag and used immunize the BALB／c mice． The specific monoclonal antibodies(mAbs)were prepared by the cell fusion technique．Western Blot was used detect specificity of mAbs．Results The prokaryotic plasmid expressing the recombinant protein was constructed，and the GP73 recombinant protein was expressed and purified．Five hybridoma cell lines that secreted anti—GP73 mAbs were obtmned．2 of 5 mAbs were the IgGl subtype．Western Blot indicated the mAbs showed specific combination with GP73 protein．Conclusion The GP73 recombinant protein is highly purified and has strong antigenicity．The anti-GP73 mAbs were prepared successfully． 【Key words】 Golgi apparatus；Protein P73； Clone molecular； Antibodies monoclonal；Liver neoplasms 原发性肝癌(primary hepatic cancer，PHC)是世 蛋白(AFP)仍然是临床上