NY-ESO-1/GST融合蛋白在大肠杆菌的表达、纯化及鉴定

Prokaryotic expression,purification and identification of NY-ESO-1/GST fusion protein in E. coli

目的:构建肿瘤-睾丸抗原NY-ESO-1与GST融合基因的原核表达载体,在大肠杆菌中表达并对融合蛋白NY-ESO-1/GST进行纯化和初步鉴定。方法:设计针对NY-ESO-1的特异性引物,采用聚合酶链反应(PCR)从睾丸组织的cDNA文库扩增NY-ESO-1基因片段,经上下游引物所引入的EcoR I和Xho I双酶切后克隆入原核表达载体pGEX-4T1中GST标签的下游,构建重组表达载体pGEX-4T1-NY-ESO-1,并将该载体转化大肠杆菌BL21(DE3)pLysS中,IPTG诱导表达NY-ESO-1/GST融合蛋白,经不同浓度尿素洗脱纯化后,表达产物通过SDS-PAGE和Western blot法进行鉴定。结果:重组质粒经限制性内切酶EcoR I和Xho I双酶切鉴定和IPTG诱导表达NY-ESO-1/GST的SDS-PAGE分析表明,表达产物的相对分子质量(Mr)为44 000,与理论值相符,并主要以包涵体形式存在,灰度扫描分析显示融合蛋白表达量占菌体蛋白总量的90%,Western blot结果证实该NY-ESO-1/GST可与抗GST单克隆抗体(mAb)发生特异性结合反应,提示为融合蛋白。结论:成功构建了NY-ESO-1基因原核表达载体pGEX-4T1-NY-ESO-1,利用大肠杆菌表达系统,获得了较高纯度的包涵体形式NY-ESO-1/GST融合蛋白。

AIM： To construct an expression plasmid for NY-ESO-1 gene and identify the expression of recombinant protein NY-ESO-1/GST in E. coil METHODS： NY-ESO-1 segment was amplified from the testis cDNA library by RT- PCR and cloned into the prokaryotic expression vector pGEX4T-1 downstream tagged by GST to construct the expression plasmid pGEX-4T1-NY-ESO-1. The recombinant vector was transformed to BL21 （DE3） and NY-ESO-1/ GST fusion protein was induced expression by IPTG. The protein was purified by urea elution and identified by SDS- PAGE and Western blotting. RESULTS： The NY-ESO-1 segment was successfully amplified and its sequence was identical with that published in GenBank. The BL21 （DE3） pLysS containing the pGEX-4Tt-NY-ESO-1expressed a Mr 44 000 fusion protein under the induction of IPTG. The purity of the protein was 90%. Western blotting proved that NY- ESO-1/GST had a specific reaction with anti-GST mAb. CONCLUSION： The prokaryotic expression vector of NY- ESO-1 has been constructed and the fusion protein NY- ESO-I/GST of high purity is successfully expressed.