人rBPI_(23)在大肠杆菌DH5α中的表达及其多克隆抗体的制备

Expression of human rBPI_(23) in E.coli DH5α and preparation of anti-rBPI_(23) polyclonal antibody

目的制备人rBPI23蛋白并免疫家兔获得特异性多克隆抗体。方法将本室制备的pBV220-synBPI600表达载体转化感受态E.coliDH5α,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鉴定分子质量,West-ernblot鉴定抗原性;免疫家兔获得抗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,Westernblot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显示其分子质量约23ku,与预期结果相符;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,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,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。

Objective To prepare human rBPI23（bactericidal/permeability increasing protein, BPI）and to develop its specific polyclonal antibody by immunizing rabbits with rBPI23. Methods The pBV220-synBPI600 expressing vectors were transferred into competent cells, and human rBPI23 was expressed as inclusion body by temperature control. Its molecular weight was identified by SDS-PAGE, and antigenic specificity was analyzed by Western-blot. The antiserum against rBPI23was developed by immunizing rabbits with rBPI23, 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 E. coli DH5α. SDS-PAGE results showed that its molecular weight is 23 ku, which is consistent with the theoretical expectation. Western-blot analysis showed that the recombinant protein could be specifically recognized by commercial rabbit polyclonal antibody against human BPI. High titer of polyclonal antibody （1：320000） was acquired by immunizing rabbits with rBPI23. 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 with rBPI23, which lay a foundation for preparation of its monoclonal antibody and later setting up immunological detecting method for BPI.