人心肌肌钙蛋白I的原核表达与纯化

Expression and purification of human cardiac troponin I

目的构建人心肌肌钙蛋白I(human cardiac 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(GeneBank M64247)公布的hcTnI基因序列一致。pQE30-hcTnI在大肠杆菌M15中经IPTG诱导后表达出相对分子量约为29 kD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。

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. Results The sequence of amplified hcTnI gene was consistent with that reported in GerrBank （M64247）. pQE30- hcTnI induced by WIG in E. coli MlJwas about 29 kD, and a special band was identified by Western blotting after Ni2 ＋ -NTA affinity chromatography. Conclusion Human cardiac troponin I gene was successfully cloned and expressed in E. coli. Highly purified hcTnI was gained by Ni2 ＋ -NTA affinity chromatography, which improved the research on anti-cTnI monoclonal antibody and establishment of quick clinical detection.