人泛素-核糖体蛋白27a原核表达载体构建及表达

Construction and prokaryotic expression of human ubiquitin-ribosomal protein S27a recombinant plasmid

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。

Objective To construct and express human ubiquitin-ribosomal protein S27a（UBRPS27a） with gl- utatione 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 fur- ther studv of its molecular structure and function and its roles in cancer develonment.