Yang Yan, Huang Huang, Zhang Zhenghua, Wang Baoju, Tian Yongjun, Lu Mengji, Yang Dongliang
Center of Experimental Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
J Huazhong Univ Sci Technolog Med Sci. 2007 Aug;27(4):411-4. doi: 10.1007/s11596-007-0415-4.
The objective of this study is to express the carbohydrate recognition domain (CRD) of the asialoglycoprotein receptor (ASGPR) H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E.coli BL21(DE3)plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemical staining (IHC). The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marmota Himalayan-for the research of infectious diseases of hepatitis viruses and liver cancer treatment.
本研究的目的是在体外表达喜马拉雅旱獭去唾液酸糖蛋白受体（ASGPR）H1和H2亚基的碳水化合物识别结构域（CRD），并制备针对重组蛋白的多克隆抗体。采用RT-PCR从喜马拉雅旱獭肝脏组织中扩增ASGPR CRDH1和CRDH2。将扩增产物亚克隆到原核表达载体pRSET-B中，并在大肠杆菌BL21(DE3)plysS中表达。用Ni-NTA旋转柱纯化重组蛋白。将纯化后的蛋白接种到BALB/c小鼠体内制备多克隆抗体。通过酶联免疫吸附测定（ELISA）、蛋白质印迹法和免疫组织化学染色（IHC）评估抗体的敏感性和特异性。多克隆抗体对变性和天然ASGPR蛋白均显示出高敏感性和特异性。我们成功扩增并表达了喜马拉雅旱獭的ASGPR CRD。喜马拉雅旱獭ASGPR CRDH1和CRDH2的核酸序列已提交至Genbank，序列ID分别为DQ 845465和DQ845466。所制备的蛋白和抗体可用于一种新的动物模型——喜马拉雅旱獭——中针对肝炎病毒传染病研究和肝癌治疗的靶向基因治疗。
