| 摘要: |
| 目的 制备人去唾液酸糖蛋白受体(ASGPR)的小鼠单克隆抗体,并应用该抗体检测ASGPR在细胞系和组织中的表达。方法 对ASGPR H1大亚基进行序列分析,选取ASGPR1全长序列制备免疫原。通过RT-PCR合成ASGPR1 cDNA,构建ASGPR1表达重组载体,在E. coli BL21中表达后纯化,获得目的抗原。应用传统融合杂交瘤技术制备小鼠单克隆抗体,常规检测单抗亚类和效价,竞争抑制实验鉴定单抗特异性。用流式细胞术和免疫组织化学法分别检测ASGPR在多种肝源性与非肝源性细胞系以及多种肝组织中的表达。结果 制备的单克隆抗体亚型为IgG1,效价达112 800。竞争抑制实验结果显示该单抗具有很好的ASGPR结合特异性,而且识别的肽段位于ASGPR胞外段。流式细胞术检测结果显示,ASGPR不同程度地表达于肝源性细胞系,但不表达于非肝源性细胞系。免疫组织化学检测结果显示,ASGPR专一性表达于正常肝组织和肝癌组织,在肝癌组织的表达与分化程度有关,高分化肝癌中的阳性率高于低分化肝癌(75.0% vs 28.6%,P<0.05)。结论 成功制备高特异性人ASGPR小鼠单克隆抗体,适用于用流式细胞术和免疫组织化学法检测ASGPR表达,可用于临床病理鉴定原发性肝癌与转移性肝癌。 |
| 关键词: 去唾液酸糖蛋白受体 单克隆抗体 肝肿瘤 流式细胞术 免疫组织化学 |
| DOI： |
| 投稿时间：2013-04-19修订日期：2013-06-28 |
| 基金项目:国家传染病重大专项课题(2012ZX10002012-010),国家高技术研究发展计划(“863”计划,2007AA02Z461),国家自然科学基金(81172207, 81272669). |
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| Preparation of mouse anti-human asialoglycoprotein receptor monoclonal antibody and its preliminary application |
| LI Jun1,2,CHEN Lei2,SUN Bin2,ZHANG Yu2,QIAN Hai-hua2,YIN Zheng-feng2* |
(1. School of Clinical Medicine, Medical College of Soochow University, Suzhou 215006, Jiangsu, China
2. Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China
*Corresponding author.) |
| Abstract: |
| Objective To prepare a mouse monoclonal antibody against human asialoglycoprotein receptor (ASGPR), and to apply it for detecting ASGPR expression in cell lines and tissues. Methods The structure of ASGPR H1 major subunit was analyzed and the full length of ASGPR1 was selected to synthesize immunizing peptide. cDNA was amplified by RT-PCR and then subcloned into prokaryotic vector pGEX-4T-1. The recombinant protein was expressed by E. coli BL21 and purified for subsequent immunization. The conventional hybridoma technique was used to generate mouse monoclonal antibody. The isotype and the titer were regularly tested. Inhibition experiment was conducted to identify the specific binding of the antibody to ASGPR. Finally, the expression of ASGPR was detected in various intra-hepatic and extra-hepatic cell lines by flow cytometry and in different liver tissues by immunohistochemistry method. Results Monoclonal antibody against human ASGPR was successfully prepared and was identified as IgG1, with the titer reaching 112 800. Inhibition experiment indicated a satisfactory specific binding of the antibody to ASGPR and that the recognition epitope was located in the extracellular domain of ASGPR. Flow cytometric analysis showed various levels of ASGPR expression in intra-hepatic cell lines, but not in extra-hepatic cell lines. Immunohistochemistry detection showed that ASGPR was specifically expressed in the normal liver tissues and hepatocellular carcinoma (HCC) tissues, and the expression in HCC tissues was associated with the differentiation degree, with the expression being significantly higher in well-differentiated HCC than that in the poorly-differentiated HCC (75.0% vs 28.6%, P<0.05). Conclusion We have successfully prepared the monoclonal antibody against human ASGPR with high specificity; the antibody can be used for flow cytometric analysis and immunohistochemistry detection of ASGPR and for clinical distinguish of primary or metastatic liver cancer. |
| Key words: asialoglycoprotein receptor monoclonal antibodies liver neoplasms flow cytometry immunohistochemistry |
