Bioinformatics of mouse uteroglobin binding protein and its polyclonal antibody preparation / 生理学报

ABSTRACT

To prepare anti-mouse uteroglobin binding protein (mUGBP) polyclonal antibody, two polypeptides were synthesized based on the bioinformatics analysis of mUGBP, and New Zealand white rabbits were immunized separately with each peptide coupled with keyhole limpet hemocyanin (KLH). The data indicate that a 13-amino acid polypeptide (positions 221st-233rd) was able to generate anti-peptide antibodies. The titer of the antisera detected with ELISA was 1:10(8). The antisera were then purified with immuno-affinity chromatography to obtain antibodies. Western blot analysis of mUGBP expressed as a fusion protein with a green fluorescent protein (GFP) was performed on the cell lysates of COS-1 cells with the purified antisera, suggesting that the antisera specifically recognized UGBP. By immunohistochemistry and indirect immunofluorescence analysis, we examined the expression of UGBP in the lung tissues from a patient undergoing surgical lung resection for a tumor and from normal mouse lung tissue, and found for the first time that UGBP protein was widely expressed in both mouse and human lung tissue with the most abundant expression in bronchial epithelial cells. These results suggest that the antigen epitopes of mUGBP are well predicted by using bioinformatics analysis. We have obtained anti-mUGBP polyclonal antibody, which will be useful for further investigation.