中文摘要：

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ASGPRH1大亚基进行序列分析，选取ASGPRl全长序列制备免疫原。通过RT-PCR合成ASGPRlcDNA，构建ASGPRl表达重组载体，在E．coliBL21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IgGl，效价达1：12800。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％VS28．6％，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。

英文摘要：

Objective To prepare a mouse monoclonal antibody against human asialoglycoprotein receptor （ASC-PR）, 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 irnmunohistocbemistry method. Results Monoclonal antibody against human ASC-PR was successfully prepared and was identified as igG1, with the titer reaching 1 ： 12 800. 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. Immunobistochemistry 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 28.6M, 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.