小鼠子宫珠蛋白结合蛋白的生物信息学分析及其多克隆抗体制备  

Bioinformatics of mouse uteroglobin binding protein and its polyclonal antibody preparation

作　　者：李晨[1,2] 刘伟[1] 汉建忠[1] 冯丹丹[1] 刘惠君[1] 罗自强[1] 

机构地区：[1]中南大学湘雅医学院生理学系,长沙410078 [2]长治医学院生理学教研室,长治046000

出　　处：《生理学报》2013年第2期217-223,共7页Acta Physiologica Sinica

基　　金：supported by the National Natural Science Foundation of China(No.30670770;30870916;81100057);Precision Instruments Sharing Foundation of Central South University;China(No.2012-02;2012-12)

摘　　要：本文旨在制备小鼠子宫珠蛋白结合蛋白(mouse uteroglo binbinding protein,mUGBP)多克隆抗体,为后续研究工作奠定基础。通过生物信息学分析方法预测mUGBP蛋白的跨膜结构、理化特性、疏水性等因素,设计出两段多肽,分别与匙孔戚血蓝蛋白(keyhole limpet hemocyanin,KLH)交联后免疫新西兰兔,结果显示其中一个含13个氨基酸残基的多肽序列(221st～233rd)可用作抗原免疫动物,并成功获得高效价的抗mUGBP多克隆抗体。通过ELISA法检测其效价为1:108,亲和层析纯化后Westernblot检测抗体特异性,并将制备的抗体应用于免疫印迹及人和小鼠肺组织免疫组织化学和免疫荧光检测,结果显示本研究制备的抗体具有特异性,且用该抗体成功在人和小鼠气道上皮细胞和肺血管内皮细胞检测到UGBP蛋白表达。结果表明,本研究通过生物信息学方法成功预测了抗原表位,并据此预测成功制备了高效价、高特异性的抗mUGBP多克隆抗体。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 cou- pled 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 1：10s. 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 rec- ognized 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 polyctonal antibody, which will be useful for further investigation.

关 键 词：子宫珠蛋白 小鼠 抗体 生物信息学 

分 类 号：R392.1[医药卫生—免疫学]