人接头蛋白NRBP的表达、纯化及多克隆抗体制备

目的:制备接头蛋白NRBP的兔多克隆抗体,并检测该抗体的效价及特异性。方法:PCR方法以重组质粒PEF-NRBP为模板,获得NRBP全长及NRBP(1-99Aa)cDNA,构建到原核表达质粒pET-21a及pGEX-6P-1中。分别转入大肠杆菌BL21菌株,IPTG诱导表达后,纯化并鉴定表达产物将其免疫家兔,间接ELISA法及免疫印迹等方法鉴定抗体。结果:成功获得人NRBP的cDNA,构建得到相关的原核表达质粒,在大肠杆菌中可诱导性高表达,纯化后蛋白免疫家兔制备得到的抗血清经ELISA检测为阳性结果,4只免疫家兔的抗体效价约为1:5 200～1:40 000。Western印迹结果显示,该抗体可特异性的识别真核细胞外源性及内源性约60kDa的NRBP蛋白,并且具有较强免疫沉淀能力。结论:NRBP多克隆抗体具有很好的特异性和敏感性,该抗体的成功制备为进一步研究NRBP的功能提供了重要工具。

Prokaryotic Expression, Purification of Human Adapter Protein NRBP and Preparation of Its Polyclonal Antibody

Objective:To prepare anti-NRBP antibody and study the titers and specificity of this antibody.Method:The full-length cDNA or cDNA encoding the first 99 Aa at N terminal of human NRBP was amplified from pEF-NRBP plasmid by PCR and then subcloned into prokaryotic expression vector pET-21a or pGEX-6P-1.The recombinant plasmids were then transformed into E.coli BL21 and induced.The fusion proteins purified were used to immune rabbit.The titers and specificity of the generated antibodies were analyzed by indirect ELISA and Western blot.Result:The human NRBP cDNA was obtained successfully.The recombinant NRBP or NRBP(1-99Aa) can be expressed by IPTG induction,the purified proteins were used to immune rabbit.The result of ELISA was positive,and the titers of antibody from four different immunized rabbits were from 1:5 120 to 1:40 000.Western blot analysis showed that the antibody can detect exogenous and endogenous NRBP protein(about 60 kDa) and had strong ability of immunoprecipitation.Conclusion:The anti-NRBP antibody was approved having good specificity and sensitivity.The polyclonal antibody of NRBP provided an important tool to study the function of NRBP.