小鼠激活素受体相互作用蛋白2的原核表达及抗血清制备

Prokaryotic Expression and Preparation of Antiserum for Mouse Activin Receptor-interacting Protein 2

[目的]克隆小鼠激活素受体相互作用蛋白2(ARIP2)cDNA序列,利用大肠杆菌表达ARIP2,制备兔抗小鼠ARIP2的多克隆抗血清。[方法]采用RT-PCR方法,将小鼠ARIP2基因克隆到pET-32a(+)质粒,构建ARIP2原核表达载体并转化到大肠杆菌,IPTG诱导融合蛋白的表达,经亲和层析纯化获得可溶性重组蛋白,免疫家兔后制备ARIP2抗血清,分别采用ELISA,Western blot检测抗体效价和特异性。[结果]测序结果表明重组质粒pET32a(+)-ARIP2构建成功;SDS-PAGE分析表明获得的重组蛋白为可溶蛋白;经过亲和层析后得到有效分离;Elisa法测定的抗血清效价为1∶50000;Western blot鉴定结果表明抗血清能与目的蛋白特异性结合。[结论]成功将ARIP2进行了原核表达并制备了高效价、高特异性的兔抗小鼠ARIP2抗血清,为进一步研究ARIP2功能提供了有力工具。

[Objective] The study aims to clone mouse activin receptor-interacting protein 2 （ARIP2） sequences and express recombinant ARIP2 E. coil, and prepare high quality rabbit polyclonal antiserum. [Method] The out-membrane fragment of ARIP2 gene is amplified by PCR from mouse liver cDNA pool, which is cloned into pET-32a（ ＋） plasmid. E coli B121 （DE3） transformed with recombinant pET-ARIP2 plasmid is induced by IFTG to express ARIP2 in high level, expressed soluble ARIP2 is purified by NI-NTA agarose to immunize rabbit. The titer and specificity of the produced antiserum are evaluated by FT：ISA and Western blot, respectively. [Result] DNA sequencing indicates the cloned ARIP2 gene is correctly inserted into the pET plasmid. The results of SDS-PAGE show that the ARIP2 fragment is mainly expressed in soluble form and can be purified by NI-NTA agarose. The titer of the rabbit ARIP2 antiserum detected by EIISA is 1：5 × 10^4, and it can specifically combine with ARIP2 in mouse liver and kidney tissues, identified by western blot. [Conclusion] The high quality ARIP2 polyclonal antiserum has been successfully prepared, which provides a sharp tool for further researching the function ARIP2.