癌-睾丸抗原GAGE-1基因的重组、表达及纯化

Recombinantion,Expression and Purifi cation of Cancer Testis Antigen GAGE-1

目的为进一步研究临床应用癌-睾丸抗原GAGE-1诱导特异性抗肿瘤免疫反应,对GAGE-1基因进行克隆、体外原核重组表达及蛋白分离纯化。方法运用RT-PCR技术从人QGY-7701肝癌细胞株中扩增GAGE-1基因片段,插入原核表达载体pGEX-6p-1中,构建重组表达质粒pGEX-6p-1-GAGE-1,并导入大肠杆菌BL21,IPTG诱导目的蛋白表达;经包涵体透析复性及GST柱亲和层析纯化目的蛋白,SDS-PAGE电泳分析鉴定。结果扩增得到GAGE-1基因5’端251 bp片段,与GeneBank公布的序列一致,构建的PGEX-6p-1-GAGE-1原核表达质粒经IPTG诱导,在大肠杆菌中表达分子量约35.7 kD的GST-GAGE-1融合蛋白,纯化后蛋白纯度为90%以上。结论成功构建PGEX-6p-1-GAGE-1重组表达质粒,并成功表达纯化了GST-GAGE-1融合蛋白,为进一步深入研究GAGE蛋白奠定了基础。

Objective To study GAGE-1 gene cloning, in vitro prokaryotic expression and protein separation and purification for future clinical application to obtain specific anti-tumor immune response induced by cancer-testis antigens GAGE-1.Methods GAGE-1 segment from QGY-7701 was amplified by RT-PCR and cloned into the expression vector pGEX-6p-1 to construct the expression plasmid pGEX-6p-1-GAGE-1. The recombinant vector was transformed to E.coli BL21 and GST fusion protein expressed was induced by IPTG. The protein was purifi ed by GST affi nity chromatography and was analyzed by SDS-PAGE. Results The sequence of 5 'terminal 251bp of GAGE-1 segment was amplified and identical with the published counterpart in GeneBank. The BL21(DE3) containing the PGEX-6p-1-GAGE-1 expressed a Mr 35.7kD GST-GAGE-1fusion protein. The purity of the protein was more than 90%.Conclusion The recombinant expression vector PGEX-6p-1-GAGE-1 was constructed successfully, and the fusion protein was expressed and purifi cated,which is the foundation for further study of GAGE protein.