Prokaryotic Expression and Purification of CD3ε Chain of Human T Lympho-cyte

Objective To express the CD3e chain of human T lymphocytes(hCD3e)in prokaryotic cells and purify the ex-pressed product.Methods hCD3e 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-hCD3e 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-hCD3e 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 hCD3e 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 CD3e antibody and goat anti-GST antibody.Conclusion GST-hCD3e fusion protein was successfully expressed in prokaryotic cells and purified.