MAGE-A3多克隆抗体的制备及在胃癌细胞检测中的应用  

Preparation of MAGE-A3 polyclonal antibody and its application in detection of human gastric cancer cell

作　　者：蔡一奇 程开[1] 陈旭东[1] 陈孝冬 岑丹维 张婵琼[2] 宋易玲 毛姗姗[2] 叶晓鲜 张丽芳[2] 朱冠保[1] 

机构地区：[1]温州医科大学附属第一医院胃肠外科,浙江温州325015 [2]温州医科大学分子病毒与免疫研究所微生物学与免疫学教研室,浙江温州325035

出　　处：《温州医科大学学报》2017年第5期318-324,共7页Journal of Wenzhou Medical University

基　　金：国家自然科学基金资助项目(81372447);浙江省自然科学基金资助项目(Y2100660)

摘　　要：目的:通过原核表达系统制备人黑色素瘤抗原A3(MAGE-A3)蛋白,制备其多克隆抗体,经鉴定后用于胃癌细胞检测。方法:将MAGE-A3全长核酸序列经原核密码子优化后全基因序列合成,克隆至原核表达载体pET21a(+),构建pET21a(+)/MAGE-A3重组质粒,转化至大肠杆菌BL21(DE3),经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达MAGE-A3蛋白,并经SDS-PAGE及Western blot分析鉴定;纯化后的MAGE-A3蛋白免疫日本大耳白兔制备多克隆血清抗体,用ELISA、Western blot、免疫荧光技术分析该多克隆抗体的特异性,并用免疫组织化学技术鉴定MAGE-A3兔多克隆抗体在人胃癌细胞免疫检测应用中的可行性。结果:成功构建重组质粒pET21a(+)/MAGE-A3,并经原核表达系统表达MAGE-A3蛋白。SDS-PAGE显示目的蛋白分子质量大小约为48 k Da,与预期蛋白大小一致,并以His单抗进行Western blot鉴定,出现了特异性的单一条带。MAGEA3蛋白免疫日本大耳白兔,可诱导兔产生特异性抗体,免疫后第8周达到高峰,经Western blot检测显示多克隆血清可特异性识别靶蛋白,出现阳性单一条带;经免疫荧光检测显示,在MAGE-A3阳性细胞A-375(黑色素瘤细胞株)和SGC-7901细胞(胃癌细胞株)的胞浆内出现荧光团块。表明兔多克隆抗体可特异性识别天然的MAGE-A3蛋白。结合免疫荧光结果后经ELISA检测,可认为该多克隆Ig G抗体效价可达1:40 000;免疫组织化学检测显示在A-375和SGC-7901肿瘤组织的胞浆内出现团块状棕黄色颗粒样沉淀,而在BGC-823肿瘤组织中呈阴性,表明制备的MAGE-A3兔多克隆抗体能够特异性地识别肿瘤组织中MAGE-A3蛋白。结论:通过原核表达系统制备了MAGE-A3蛋白,并免疫兔制备了MAGE-A3蛋白特异性兔多克隆抗体,同时可以特异性地识别人胃癌细胞中的MAGE-A3蛋白,可作为免疫诊断用抗原。Objective： To prepare Human MAGE-A3 （MAGE-A3） protein by prokaryotic expression sys-tem. At the same time, a polyclonal antibody was prepared to against it, after preliminary identifcation and ap-plication. Methods： The MAGE-A3 full-length nucleic acid sequences were synthetized and cloned into pET21a （＋） plasmid to construct pET21a （＋）/MAGE-A3 restructuring plasmids. After sequencing, the recombinant plas-mid was transformed into E.coli BL21 （DE3）. The MAGE-A3 protein was expressed through Isopropyl β-D-1-thiogalactopyranoside （IPTG） inducing. Then the recombinant protein of MAGE-A3 was purifed by Ni-NTA affnity chromatography and confrmed by SDS-PAGE and Western blot analysis, the rabbits were immunized with the purifed protein MAGE-A3 in order to prepare the polyclonal antibody. The titers of specifc antibodies were detected by ELISA. The specifcity activity was further detected by Western blot and immunofuorescence technique analysis. Its preliminary application was detected in human gastric cancer cells by immunohisto-chemical techniques. Results： The recombinant plasmids pET21a（＋）/MAGE-A3 （full-length） were successfullysystem. SDS-PAGE showed the size of the protein about 48 kDa was consistent with the expected size of the pro-tein and identifed by Western blot. The polyclonal antibodies were produced in the rabbits immunized with the MAGE-A3 recombinant protein. The Antigen-antibody reactions reached a peak in the eighth week. Western blot analysis indicated that the polyclonal antibody could specifcally recognize the MAGE-A3 recombinant protein. The immunofuorescence showed that fuorescent clumps appeared in intracytoplasm of MAGE-A3 positive cells A-375 （melanoma cell line） and SGC-7901 cells （gastric cancer cell line）. It confrmed that polyclonal antibody could specifically recognizes natural MAGE-A3 protein. ELISA analysis indicated the titer of the polyclonal IgG was up to 1：40 000 in the eighth week; Immunohistochemistry display appeared in the cytop

关 键 词：人黑色素瘤抗原A3 原核表达系统 多克隆抗体 胃肿瘤 

分 类 号：R730.3[医药卫生—肿瘤]