原癌基因Pokemon的克隆原核表达及纯化

Cloning,prokaryotic expression and purification of proto-oncogene Pokemon

目的克隆小鼠Pokemon(POKeryhroidmyeloidontogenicfactor)基因并进行原核表达及重组蛋白的纯化。方法应用反转录聚合酶链反应(RT-PCR)扩增目的基因片段,克隆人pGEM-T-easy载体,经鉴定后,以限制性核酸内切酶分别消化重组质粒及原核表达载体pET-30a(+),体外定向连接,进行PCR、内切酶酶切及DNA序列分析,阳性重组表达质粒进一步转化到大肠杆菌E.coliBL21(DE3)中诱导表达,经Ni-NTA亲和层析纯化融合蛋白,Westernblotting检测其特异性。结果限制性核酸酶切及序列分析表明重组表达质粒包含Pokemon基因编码区,阅读框架无移位;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)显示有预期相对分子质量为36000的重组蛋白表达,经亲和层析纯化后,融合蛋白的纯度达到90%以上;Westernblotting印迹表明纯化的融合蛋白具有特异的免疫反应特性。结论采用基因克隆技术成功构建了Pokemon原核表达载体,并获得了高纯度的重组蛋白,为后期抗体的制备及进一步研究该基因与肿瘤发生的关系奠定了基础。

Objective To clone mouse Pokemon gene and to induce its expression in E.coli,further to purify the recombinant protein. Methods Target gene was amplified by reverse transcription polymerase chain reaction（RTPCR） and cloned into pGEM-T-easy.After being identified,the recombinant plasmid was digested and cloned into prokaryotic expression vector pET-30a（＋） which was digested with the same restriction enzymes.PCR, retriction enzyme digestion and direct sequencing were used to identify the positive recombinant expression plasmid which was transformed into the E.coli BI21 （DE3） and induced to express recombinant protein.The fusion protein was further purified with Ni-NTA affinity chromatography and confirmed with Western blotting. Results Restriction enzyme digestion and DNA sequencing suggested that the recombinant expression plasmid contained correct encoding region of Pokemon, without reading frame shift.SDS-PAGE demonstrated that the recombinant protein was highly expressed with the expected molecular weight at 36 000 and the purity of the fusion protein was above 90% after being purified with affinity chromatography.Western blotting also demonstrated the immune specificity of the purified fusion protein. Conclusion The prokaryotic expression vector of Pokemon is successfully constructed by gene cloning technique,and its recombinant protein with high purity was obtained.These results may play basic roles for antibody preparation and further study into the relationship between Pokemon and tumorigenesis.