【摘要】 目的构建并表达携带谷胱甘肽S-转移酶(GST)标签的人泛素-核糖体蛋白S27a(UBRPS27a)原核表达载体。方法利用反转录聚合酶链反应从HL-60细胞中扩增RPS27A基因全长,克隆至pMD-19T载体中,酶切回收后插入原核表达载体pGEX4T-1,构建重组表达载体pGEX4T1-RPS27A,转化大肠埃希菌BL21,以异丙基-β-D-硫代半乳糖苷诱导表达GST-UBRPS27a融合蛋白。结果经测序与酶切鉴定,重组质粒pGEX4T1-RPS27A构建正确;经重组质粒转化的BL21细菌诱导4h后可高效表达UBRPS27a融合蛋白。结论成功构建UBRPS27a原核表达载体,为进一步研究其结构、功能及其在肿瘤发生发展中的作用奠定了基础。更多还原

【Abstract】 Objective To construct and express human ubiquitin-ribosomal protein S27a(UBRPS27a) with glutatione S-transferase(GST) tag.Methods RPS27A cDNA,obtained by reverse transcript-polymerase chain reaction from HL-60 cells,was inserted into pMD-19T vector,which then was digested by restriction endonuclease and cloned into pGEX4T-1 vector to construct recombinant plasmid pGEX4T1-RPS27A.BL21 E.coli was then transformed with pGEX4T1-RPS27A recombinant plasmid and induced to express GST-UBPRS27a protein.Results The recombinant plasmid pGEX4T1-RPS27A was successfully constructed according to double digestion and DNA sequencing.The soluble GST-UBRPS27a fusion protein was induced with high expression efficiency.Conclusion The construction of RPS27A recombinant vector and prokaryotic expression of the UBRPS27a protein would lay the foundation for further study of its molecular structure and function and its roles in cancer development.更多还原