重组人细胞角蛋白9 cDNA的原核表达及纯化

Prokaryotic expression and purification of the cDNA of recombinant human cytokeratin 9

目的克隆人细胞角蛋白CK9的cDNA,通过原核表达系统表达融合蛋白并进行纯化和鉴定。方法从人角质形成细胞系(HaCaT)中提取RNA,以RT-PCR反转录cDNA,使用特异性引物PCR扩增出人CK9编码区全长并克隆到pET-28a载体上,再用IPTG诱导融合蛋白his-CK9的表达。表达的融合蛋白通过Ni 2+亲和层析纯化后,进行SDS-PAGE和Western blot鉴定。结果测序鉴定构建表达载体中CK9的cDNA序列正确;融合蛋白his-CK9可在大肠杆菌中诱导表达;纯化的融合蛋白his-CK9纯度较高;纯化的融合蛋白his-CK9可与商品化CK9抗体特异性结合。结论成功构建原核表达载体pET-28a-CK9,并成功诱导及纯化融合蛋白his-CK9。

Objective To clone and fuse the cDNA of human cytokeratin 9 in prokaryotic expression system, and purify and identify the fusion protein. Methods The cDNA fragment of human cytokeratin 9 was amplified from human keratinocyte （HaCaT） total RNA with specific primers. The PCR products were cloned into vector pET-28a, then the fusion protein of his-CK9 was induced by IPTG. The expressed fusion protein of his-CK9 was purified by nickel ion affinity chromatography and identified by SDS-PAGE and Western blot. Results Thesequencing proved that the recombinant vector of the cDNA of CK9 was correct. The fusion protein of his-CK9 was induced to be expressed in E. coli. The fusion protein of his-CK9 was highly purified and his-CK9 showed specific binding to the commercialized antibodies of CKg. Conclusion The recombinant vector of pET-28a-CK9 has been successfully constructed, and the fusion protein of his-CK9 has been successfully expressed and purified.