%0 Journal Article
%T 重组人细胞角蛋白9 cDNA的原核表达及纯化<br>Prokaryotic expression and purification of the cDNA of recombinant human cytokeratin 9
%A 王
%A 博
%A 周
%A 艳
%A 侯卫坤
%A 蔡永松
%A 张
%A 英
%A 王林玉
%A 韩
%A 燕
%A 孟列素
%J 西安交通大学学报(医学版)
%D 2017
%R 10.7652/jdyxb201704007
%X 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。<br>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
%K 原核表达系统
%K 细胞角蛋白9
%K 融合蛋白
%K 细胞骨架&lt
%K br&gt
%K prokaryotic expression system
%K cytokeratin 9
%K fusion protein
%K cytoskeleton
%U http://yxxb.xjtu.edu.cn//oa/darticle.aspx?type=view&id=201704007