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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 the polyclonal antibody of human melanoma A3 (MAGE-A3) protein by prokaryotic expression system and identify it for detection of gastric cancer cells. Methods: The full-length MAGE-A3 cDNA sequence was synthesized by digestion with full-length codon-optimized gene and cloned into prokaryotic expression vector pET21a (+). The recombinant plasmid pET21a (+) / MAGE-A3 was transformed into E.coli BL21 ), MAGE-A3 protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and identified by SDS-PAGE and Western blot analysis. The purified MAGE-A3 protein was used to immunize Japanese white rabbits The polyclonal antibody was prepared and the specificity of the polyclonal antibody was analyzed by ELISA, Western blot and immunofluorescence. Immunohistochemistry was used to identify the feasibility of polyclonal antibody against MAGE-A3 rabbit polyclonal antibody in the detection of human gastric cancer cells. Results: The recombinant plasmid pET21a (+) / MAGE-A3 was successfully constructed and expressed MAGE-A3 protein by prokaryotic expression system. SDS-PAGE showed that the molecular mass of the target protein was about 48 kDa, which was consistent with the expected protein size. Western blot analysis using His monoclonal antibody showed a single band. MAGEA3 protein was used to immunize Japanese white rabbits to induce specific antibody production in rabbits. After immunization, the peak was reached at the 8th week after immunization. The result of Western blot showed that the polyclonal serum could specifically recognize the target protein with a positive single band. After immunofluorescence Showed that a fluorophore appeared in the cytoplasm of MAGE-A3 positive cells A-375 (melanoma cell line) and SGC-7901 cells (gastric cancer cell line). This indicates that the rabbit polyclonal antibody specifically recognizes the native MAGE-A3 protein. The result of immunofluorescence showed that the titer of the polyclonal Ig G antibody was 1: 40 000 by ELISA. Immunohistochemistry showed that there was lumpy brown in cytoplasm of A-375 and SGC-7901 tumor tissues Yellow particles precipitate, but negative in BGC-823 tumor tissue, indicating that the prepared MAGE-A3 rabbit polyclonal antibody can specifically recognize MAGE-A3 protein in tumor tissue. CONCLUSION: MAGE-A3 protein was prepared by prokaryotic expression system and MAGE-A3 protein-specific rabbit polyclonal antibody was prepared by immunization. MAGE-A3 protein was identified specifically in human gastric cancer cells and could be used as immunoassay antigen.