人心肌肌钙蛋白I的原核表达与纯化

目的构建人心肌肌钙蛋白Ⅰ（human cmdiac troponin I,hcTnI）原核表达载体并表达、分离纯化重组蛋白。方法RT-PCR从人心肌组织中克隆出hcTnI的cDNA序列构建到原核表达载体pQE30，获得重组表达质粒pQE30-hcTnI，并在大肠杆菌M15中诱导表达。镍凝胶亲和层析纯化目的蛋白，Western Blot杂交鉴定重组蛋白。结果经测序证实，获得的目的基因与（GeneBankM64247）公布的hcTnI基因序列一致。pQE30-hcTnI在大肠杆菌M15中经WFG诱导后表达出相对分子量约为29kD的目的蛋白，镍柱纯化后经抗hcTnI单克隆抗体进行Western印记，在相对分子量约29kD处可见特异性着色带。结论体外成功诱导了重组hcTnI蛋白表达，通过镍凝胶亲和层析法获得纯度较高的hcTnI蛋白，为进一步获得hcTnI的抗体及建立相应的临床快速检测方法奠定了实验基础。

Expression and purification of human cardiac troponin I

Objective To construct prokaryotic plasmid of hcTnI and obtain recombinant hcTnI by prokaryotic expression and purification.Methods The encoding sequence for mature peptide of human cardiac troponin I was amplified with RT-PCR and inserted into pQE30 vector to establish the prokaryotic expressing system.The recombinant expression plasmid pQE30-hcTnI was constructed and expressed in E.coli and hcTnI was purified by Ni2+-NTA affinity chromatography.Western Blot was used to determine the recombinant hcTnI.Resu...