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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

Abstract

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.

Key words: adiponectin receptor-1;Mouse;antibody;computational biology

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Citing This Article：

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).