人rBPI23在E.coli DH5α中的表达及其多克隆抗体的制备

基础医学与临床 ›› 2007, Vol. 27 ›› Issue (11): 1219-1222.

人rBPI23在E.coli DH5α中的表达及其多克隆抗体的制备

龙军李蕴陈艳秋李丽吕喆安云庆

首都医科大学基础医学院免疫系首都医科大学首都医科大学湖北民族学院附属医院妇产科首都医科大学首都医科大学

收稿日期:2006-09-13 修回日期:2007-03-08 出版日期:2007-11-25 发布日期:2007-11-25
通讯作者: 龙军

Expression of Human rBPI23 in E.coli DH5α and Preparation of its Polyclonal Antibody

Received:2006-09-13 Revised:2007-03-08 Online:2007-11-25 Published:2007-11-25

摘要/Abstract

摘要： 摘要：目的　制备人rBPI23，免疫家兔获得特异性多克隆抗体，为制备单克隆抗体及后期建立BPI免疫学检测方法奠定基础。方法　将PBV220-synBPI600表达载体转化感受态E.coli DH5α，温控诱导后，获得以包涵体形式表达的目的重组蛋白；用SDS-PAGE鉴定分子量，Western-blot鉴定抗原性；免疫家兔获得rBPI23抗血清，经饱和硫酸铵沉淀获得多克隆抗体，间接法ELISA检测抗体效价，Western-blot分析抗体的特异性。结果　重组蛋白主要以包涵体形式表达，SDS-PAGE显示其分子量约23KD，与预期结果相符；Western-blot结果显示重组蛋白能与市售兔抗人BPI抗体特异性结合；免疫家兔获得高效价（1:320000）抗血清，Western-blot证实上述抗体能与rBPI23及人BPI标准品特异性结合。结论　成功制备了人rBPI23，免疫家兔获得高效价抗人rBPI23多克隆抗体。

关键词: rBPI23, 原核表达, 多克隆抗体制备

Abstract: ABSTRACT: Objective To prepare human rBPI23 and acquire its specific polyclonal antibody by immunizing rabbits, so that lay the foundation for preparation of its monoclonal antibody and later setting up immunological testing method for BPI. Methods PBV220-synBPI600 expressing vectors were transferred into competent cell, and the recombinant protein was expressed as inclusion body by temperature control. Its molecular weight was certified by SDS-PAGE, and antigenic specificity was analyzed by Western-blot. Antiserum against rBPI23 was acquired by immunizing rabbits, and the polyclonal antibody was purified by saturated ammonium sulphate precipitation. The titer of antibody was tested by indirect ELISA , and the specificity of antibody was analyzed by Western-blot. Results The recombinant protein was expressed mainly as inclusion body in Ecoli DH5α, SDS-PAGE showed that its molecular weight is 23 kD, which is consistent with the previous theoretical expection. The recombinant protein was analyzed by western-blot with rabbit polyclonal antibody against human BPI, and found that it could be recognized specifically by this polyclonal antibody. High titer of polyclonal antibody（1:320000） was acquired by immunizing rabbits, western-blot analysis revealed that these polyclonal antibodies could specifically react with rBPI23 and human standard sample of BPI. Conclusion Human rBPI23 was successfully prepared, and high titer of polyclonal antibody against human rBPI23 was acquired by immunizing rabbits.

Key words: rBPI23, prokaryotic expression, polyclonal antibody preparation

龙军李蕴陈艳秋李丽吕喆安云庆. 人rBPI23在E.coli DH5α中的表达及其多克隆抗体的制备[J]. 基础医学与临床, 2007, 27(11): 1219-1222.

. Expression of Human rBPI23 in E.coli DH5α and Preparation of its Polyclonal Antibody[J]. Basic & Clinical Medicine, 2007, 27(11): 1219-1222.

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