[Bioinformatics analysis of mouse adiponectin receptor-1 and its antibody preparation].
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 trans-membrane 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.
Xu
Yan-Wu
YW
Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001, China.
Yan
Li
L
Liu
Hui
H
Wu
Ye
Y
Wu
Bo-Wei
BW
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000918:Antibody Specificity; D015153:Blotting, Western; D019295:Computational Biology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D051379:Mice; D010455:Peptides; D051381:Rats; D054419:Receptors, Adiponectin / Q000737:chemistry* / Q000276:immunology
D000906:Antibodies; D000939:Epitopes; D010455:Peptides; D054419:Receptors, Adiponectin; C475419:adiponectin receptor 1, mouse