人T淋巴细胞CD3ε链的原核表达及纯化

目的原核表达并纯化人T淋巴细胞CD3ε(hCD3ε)链。方法以健康成人外周血单个核细胞总RNA为模板,采用RT-PCR法扩增人CD3ε链基因,并克隆入pCR-II载体,酶切鉴定及测序分析后,再定向克隆至原核表达载体pGEX-4T-3中,构建重组表达质粒pGEX-4T-3-hCD3ε,转化E.coli BL21(DE3),IPTG诱导表达。表达产物经GST亲和层析纯化后,进行Western blot鉴定。结果酶切分析及DNA测序证实,hCD3ε链基因被正确克隆入pGEX-4T-3载体,并能在原核系统中稳定表达。表达产物的相对分子质量为49 500,0.2 mmol/L IPTG 25℃诱导表达4.5 h,目的蛋白的表达量最高,占菌体总蛋白的29.3%,其中可溶性表达占12.8%。纯化的融合蛋白纯度可达86.1%,可分别被兔抗人CD3ε多抗和羊抗GST多抗识别。结论已成功地原核表达并纯化了GST-hCD3ε融合蛋白。

Prokaryotic Expression and Purification of CD3e Chain of Human T Lymphocyte

Objective To express the CD3ε chain of human T lymphocytes(hCD3ε)in prokaryotic cells and purify the ex-pressed product.Methods hCD3ε gene was amplified by RT-PCR using the total RNA of healthy human PBMCs as a template and cloned into vector pCR-Ⅱ.The constructed recombinant plasmid pCR-Ⅱ-hCD3ε was identified by restriction analysis and sequenc-ing,then directionally cloned into prokaryotic expression vector pGEX-4T-3,and the constructed recombinant plasmid pGEX-4T-3-hCD3ε was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed product was purified by GST affinity chromatography and identified by Western blot.Results Both restriction analysis and DNA sequencing proved that hCD3ε gene was successfully cloned into vector pGEX-4T-3 and expressed stably in prokaryotic cells.The relative molecular mass of ex-pressed product was 49 500.After induction with 0.2 mmol / L IPTG at 25℃ for 4.5 h,the expression level of target protein reached a peak value of 29.3% of total somatic protein.A portion of 12.8% of total somatic protein was expressed in a soluble form.The ex-pressed fusion protein reached a purity of 86.1% after purification,and was recognized with rabbit anti-human CD3ε antibody and goat anti-GST antibody.Conclusion GST-hCD3ε fusion protein was successfully expressed in prokaryotic cells and purified.