NY-ESO-1和热休克蛋白70融合基因的构建和原核表达

Construction and Prokaryotic Expression of Fusion Gene of NY-ESO-1 and Heat Shock Protein 70

目的构建热休克蛋白(HSP)70和肿瘤睾丸抗原NY-ESO-1融合基因,并在大肠杆菌中诱导表达。方法应用逆转录聚合酶链反应(RT-PCR)技术,从人肝癌细胞株HepG2扩增HSP70基因,测序后插入pGEX-ESO1质粒中,构建NY-ESO-1与HSP70的N端融合基因原核表达质粒pGEX-ESO1-HSP70,IPTG诱导含有该质粒的大肠杆菌BL21(DE3)表达目的蛋白,Western blot鉴定蛋白的特异性。结果扩增得到长度为1 917 bp的人HSP70全长基因,测序结果表明与GenBank公布的序列一致;获得pGEX-ESO1-HSP70融合基因原核表达质粒,经IPTG诱导,在BL21(DE3)中表达分子量约106 kD的融合蛋白(包括GST 26 kD,NY-ESO1 10 kD,HSP7070 kD),Western blot检测该蛋白可与HSP70特异性结合。结论成功构建pGEX-ESO1-HSP70重组表达质粒,并获得高效表达的ESO1-HSP70融合蛋白,为HSP70-多肽疫苗在肿瘤免疫治疗中的作用奠定基础。

Objective To construct the fusion gene of human NY-ESO-1 and heat shock protein 70(HSP 70) and express the fusion protein in E.coli. Methods The HSP 70 gene was amplified by RT-PCR from HepG2 cell line.After sequencing, HSP70 gene was cloned into the pGEX-ESO1 vector to construct the fusion gene plasmid pGEX-ESO1-HSP70.The BL21(DE3) containing pGEX-ESO1-HSP70 was induced by IPTG.The fusion protein was identified by Western blot.Results The full-length HSP70 gene was amplified from HepG2 cell line,which sequence was identical with that reported in Genbank.The fusion gene plasmid pGEXESO1-HSP70 was constructed.The BL21(DE3) containing the pGEX-ESO1-HSP70 was induced to express a Mr 106 kD fusion protein,which could specifically recognize HSP70 antibody by Western blot.Conclusion The fusion gene plasmid of human NY-ESO-1 and HSP70 was constructed and the fusion protein was expressed successfully.It lays the foundation for HSP-peptide complex vaccines in tumor immunotherapy.