人GPC3基因的原核表达及多克隆抗体的制备

doi:10.13560/j.cnki.biotech.bull.1985.2017-0379

生物技术通报 ›› 2017, Vol. 33 ›› Issue (11): 188-193.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0379

人GPC3基因的原核表达及多克隆抗体的制备

腾桥, 夏丽洁, 张富春

新疆大学生命科学与技术学院生物资源基因工程重点实验室,乌鲁木齐 830046

收稿日期:2017-05-12 出版日期:2017-11-26 发布日期:2017-11-22
作者简介:腾桥,女,硕士研究生,研究方向：分子免疫学;E-mail：851566931@qq.com
基金资助:
国家自然科学基金资助项目（31500752）,新疆大学博士启动基金资助项目（BS150241）

Prokaryotic Expression of Human GPC3 and Preparation of Polyclonal Antibody

TENG Qiao, XIA Li-jie, ZHANG Fu-chun

Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technology,Xinjiang University,Urumqi 830046

Received:2017-05-12 Published:2017-11-26 Online:2017-11-22

摘要/Abstract

摘要： 旨在构建人磷酯酰肌醇蛋白聚糖3（Glypican3,GPC3）原核蛋白,制备小鼠GPC3抗血清。扩增人 GPC3基因cDNA的完整的开放阅读框,克隆至pET28a原核表达载体,在大肠杆菌BL21表达菌株中诱导表达磷酯酰肌醇蛋白聚糖融合蛋白,利用亲和层析的方法纯化his-融合蛋白,通过SDS-PAGE电泳检测蛋白的纯度,并测定蛋白含量;免疫Balb/c小鼠,制备抗血清。结果显示,成功制备了小鼠抗GPC3多克隆抗体,抗体滴度为1∶51 200,经Western blot检查特异性良好。原核表达的重组人GPC3蛋白具有较好的免疫原性。

关键词: 磷酯酰肌醇蛋白聚糖3, 融合蛋白, 多克隆抗体

Abstract: This work aims to construct the prokaryotic protein of glypican3（GPC3）and to prepare its anti-serum to mouse. The complete open reading frame（ORF）of human GPC3’s cDNA was amplified by PCR,and then cloned into prokaryotic expression vector of pET28a. Further,the GPC3 fusion protein was expressed in Escherichia coli BL21,the fusion his-protein was purified by affinity chromatography and the fusion protein of purity was measured by SDS-PAGE electrophoresis. Finally,the protein content was also measured. The Balb/c mice were immunized by the purified protein for preparing the anti-serum,and the anti-serum was identified with ELISA analysis. Results showed that the polyclonal antibody against GPC3 protein was successfully prepared and the titer of antisera was 1：51200;moreover,the specificity was validated to be fine by Western blot. Conclusively,recombinant human GPC3 protein expressed in the prokaryotic system has strong immunogenicity.

Key words: glypican3, fusion protein, polyclonal antibody

腾桥, 夏丽洁, 张富春. 人GPC3基因的原核表达及多克隆抗体的制备[J]. 生物技术通报, 2017, 33(11): 188-193.

TENG Qiao, XIA Li-jie, ZHANG Fu-chun. Prokaryotic Expression of Human GPC3 and Preparation of Polyclonal Antibody[J]. Biotechnology Bulletin, 2017, 33(11): 188-193.

参考文献

[1] Forner A, Llovet J, Bruix J. Hepatocellular carcinoma[J]. Lancet, 2012, 379（9822）：1245-1255.
[2] Cao H, Phan H, Yang L. Improved chemotherapy for hepatocellular carcinoma[J]. Anticancer Res, 2012, 32（4）：1379-1386.
[3] Sersté T, Barrau V, Ozenne V, et al. Accuracy and disagreement of computed tomography and magnetic resonance imaging for the diagnosis of small hepatocellular carcinoma and dysplastic nodules：role of biopsy[J]. Hepatology, 2012, 55（3）：800-806.
[4] Haruyama Y, Kataoka H. Glypican-3 is a prognostic factor and an immunotherapeutic target in hepatocellular carcinoma[J]. World J Gastroenterol, 2016, 22（1）：275-283.
[5] Tsuchiya N, Sawada Y, Nakatsura T. Biomarkers for the early diagnosis of hepatocellular carcinoma[J]. World Journal of Gastroenterology, 2015, 21（37）：10573-10583.
[6] Sung YK, Hwang SY, Park MK, et al. Glypican-3 is overexpressed in human hepatocellular carcinoma[J]. Cancer Sci, 2003, 94：259-262.
[7] Hippo Y, Watanabe K, Watanabe A, et al. Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma[J]. Cancer Res, 2004, 64：2418-2423.
[8] Qin ZN, Wang J, Wang Y, et al. Identifcation of a glypican-3-binding peptide for in vivo non-invasive human hepatocellular carcinoma detection[J]. Macromolecular Bioscience, 2017. doi:10.1002/mabi.201600335.
[9] Luo L, Wang YZ, Huang Z, et al. Signifcance of Glypican-3（GPC3）expression in hepatocellular cancer diagnosis[J]. Med Sci Monit, 2017, 23：850-855.
[10] Zhang JW, Li Y, Zeng XC, et al. miR-630 overexpression in hepatocellular carcinoma tissues is positively correlated with alpha-fetoprotein[J]. Med Sci Monit, 2015, 21：667-673.
[11] Cai L, Rao XH, Qi SU, et al. Diagnostic value of Joint Detection of GP73 and AFP-L3 in primary hepatic carcinoma with low concentration of AFP[J]. Journal of International Translational Medicine, 2015, 3：28-32.
[12] Attallah AM, El-Far M, Omran MM, et al. GPC-HCC model：a combination of glybican-3 with other routine parameters improves the diagnostic efficacy in hepatocellular carcinoma[J]. Tumor Biol, 2016, 37：12571-12577.
[13] Kim H, Xu GL, Borczuk AC, et al. The heparan sulfate proteoglycan GPC3 is a potential lung tumor suppressor[J]. American Journal of Respiratory Cell & Molecular Biology, 2003, 29（6）：694-701.
[14] Wu YL, Liu H, Ding HG. GPC3 in hepatocellular carcinoma：current perspectives[J]. Journal of Hepatocellular Carcinoma, 2016, 3：63-67.
[15] Chen CL, Huang XM, Chen CS, et al. Can glypican-3 be a disease-specifcbiomarker?[J]. Clin Trans Med, 2017, 6（18）：1-6.
[16] J. 萨姆布鲁克, D. W拉塞尔, 黄培堂等译. 分子克隆实验指南[M]. 第3版. 科学出版社, 2002.

人GPC3基因的原核表达及多克隆抗体的制备

Prokaryotic Expression of Human GPC3 and Preparation of Polyclonal Antibody

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