许言午, 闫莉, 刘慧, 武烨, 吴博威
山西医科大学生物化学与分子生物学教研室.山西,太原 030001；山西医科大学生理学教研室.山西,太原 030001
该文旨在根据小鼠脂联素受体--1(adiponectin receptor--1,AdipoR--1)的分子结构,制备抗AdipoR--1的多克隆抗体,为AdipoR--1的进一步研究创造条件。利用生物信息学分析预测小鼠AdipoR--1的理化特性、跨膜结构和抗原表位,结合AdipoR--1和AdipoR--2氨基酸序列相似性比对结果,设计出一段由16个氨基酸组成的多肽,用此肽段免疫大鼠得到多克隆抗体,利用ELISA、Western blot等方法鉴定其特异性和滴度,并将该抗体用于正常及高胆固醇血症小鼠肌肉组织AdipoR--1的表达检测。结果表明,此抗体效价较高、特异性强。以上结果提示,生物信息学分析能较好地预测小鼠AdipoR--1的抗原表位,并成功制备高特异性的多克隆抗体。
Bioinformatics analysis of mouse adiponectin receptor--1 and its antibody preparation
Xu Yanwu, Yan Li, Liu Hui, Wu Ye, Wu Bowei
Department of Biochemistry and Molecular Biology,Shanxi Medical University.Taiyuan 030001,Shanxi；China
To establish a method for preparation of anti-mouse adiponectin receptor-1 （AdipoR-1） polyclonal antibody, the polypeptide antigen corresponding to AdipoR-1 was designed by bioinformatics analysis. The possible physicochemical property and transmembrane structure were predicted by ExPASy and TMHMM, respectively. The antigen epitopes of mouse AdipoR-1 and its immunogenicity were analyzed by Antigenic Prediction and AntigenProfiler, respectively. According to the similarity analysis between AdipoR-1 and AdipoR-2 by Clustal W, a 16-amino acid polypeptide was designed as the antigen corresponding to AdipoR-1. To ensure the specificity of the polypeptide antigen, similarity search was run in the protein databases such as SWISS-PROT, PDB and Prosite databases. The polypeptide synthesized by solid-phase synthesis was used as immunogen to immunize rats to obtain anti-mouse AdipoR-1 polyclonal antibodies, the specificity and titer of which was identified by Western blot and indirect ELISA. The antibodies were applied to detect the AdipoR-1 expression in the muscle tissue in normal and cholesterolemic mice. The results from bioinformatics analysis showed that the similarity of amino acid sequences between AdipoR-1 and AdipoR-2 in mouse was 66%, and the designed polypeptide antigen corresponding to AdipoR-1 exhibited excellent immunogenicity （score=3.1）. Using the polypeptide as antigen for immunization, anti-mouse AdipoR-1 polyclonal antibodies with high titer and good specificity were obtained. The results of Western blot demonstrated that there was no statistical difference in AdipoR-1 expression in muscle tissue between normal （1.80±0.06） and cholesterolemic mice （1.71±0.11）. These results suggest that the antigen epitopes of mouse AdipoR-1 are well predicted by bioinformatics analysis, and successful preparation of the specific anti-AdipoR-1 polyclonal antibodies provides a useful tool for identification and further functional study of AdipoR-1.
许言午, 闫莉, 刘慧, 武烨, 吴博威. 小鼠脂联素受体--1的生物信息学分析及其抗体制备[J]. 生理学报 2008; 60 (1): 156-160.
Xu Yanwu, Yan Li, Liu Hui, Wu Ye, Wu Bowei. Bioinformatics analysis of mouse adiponectin receptor--1 and its antibody preparation. Acta Physiol Sin 2008; 60 (1): 156-160 (in Chinese with English abstract).