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- 2017
重组人细胞角蛋白9 cDNA的原核表达及纯化
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Abstract:
摘要：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Ni2+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。
ABSTRACT: 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 The sequencing 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 CK9. 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
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