MAGE-A3多克隆抗体的制备及在胃癌细胞检测中的应用

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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 kDa，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。MAGE-A3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IgG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。

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	蔡一奇
	程开
	陈旭东
	陈孝冬
	岑丹维
	张婵琼
	宋易玲
	毛姗姗
	叶晓鲜
	张丽芳
	朱冠保

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

Abstract：Objective: To prepare Human MAGE-A3 (MAGE-A3) protein by prokaryotic expression system. At the same time, a polyclonal antibody was prepared to against it, after preliminary identification and application. 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 plasmid 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 purified by Ni-NTA affinity chromatography and confirmed by SDS-PAGE and Western blot analysis, the rabbits were immunized with the purified protein MAGE-A3 in order to prepare the polyclonal antibody. The titers of specific antibodies were detected by ELISA. The specificity activity was further detected by Western blot and immunofluorescence technique analysis. Its preliminary application was detected in human gastric cancer cells by immunohistochemical techniques. Results: The recombinant plasmids pET21a(+)/MAGE-A3 (full-length) were successfully constructed. The MAGE-A3 recombinant protein could be expressed and purified in the prokaryotic expression system. SDS-PAGE showed the size of the protein about 48 kDa was consistent with the expected size of the protein and identified 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 specifically recognize the MAGE-A3 recombinant protein. The immunofluorescence showed that fluorescent clumps appeared in intracytoplasm of MAGE-A3 positive cells A-375 (melanoma cell line) and SGC-7901 cells (gastric cancer cell line). It confirmed 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 cytoplasm A-375 and SGC-7901 tumor tissue lumpy brown granular precipitate, while a negative in BGC-823 tumor tissues, suggesting that the preparation of the MAGE-A3 rabbit polyclonal antibody was capable identifing MAGE-A3 protein specifically in tumor tissue. Conclusion: MAGE-A3 recombinant protein is expressed by the prokaryotic expression system and its polyclonal antibody is prepared successfully. And the polyclonal antibody can specifically recognize MAGE-A3 protein in human gastric cancer cells.

Key words： human melanoma antigen-A3 prokaryotic expression system polyclonal antibody stomach neoplasms

收稿日期: 2016-06-22

基金资助: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）。

通讯作者: 朱冠保，教授，硕士生导师，Email：zgbwmc@126.com。

作者简介: 蔡一奇（1990-），男，浙江温州人，硕士生。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2017/V47/I5/318

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