重组原核表达载体pGEX-4T-1-eIF5A的构建及表达

目的构建人eIF5A基因的融合表达载体并在大肠杆菌中诱导表达pGEX-4T-1-eIF5A融合蛋白。方法以pCMV6-XL4-eIF5A作为模板,经PCR技术扩增目的片段,将获得的目的片段重组到原核表达载体pGEX-4T-1上,经酶切鉴定、DNA测序检测插入序列的正确性。将测序正确的重组表达质粒pGEX-4T-1-eIF5A转化到E.coliBL21内,经IPTG诱导后,GSTpull-down后SDS-PAGE电泳分析以及Western blot检测人eIF5A的表达。结果成功构建重组表达质粒pGEX-4T-1-eIF5A,经DNA测序证实插入序列与设计完全一致,GSTpull-down后电泳分析以及Westernblot结果说明大肠杆菌BL21诱导后表达出人eIF5A的glutathioneS-transferase(GST)融合蛋白。功能研究初步证明重组eIF5A在细胞增殖过程中发挥重要作用。结论构建成功的重组原核表达载体能在E.coliBL21内表达eIF5A的GST融合蛋白,为进一步研究人eIF5A蛋白的结构和生理功能如促进创伤修复提供帮助。

Construction and Expression of Recombinant Prokaryofic Expression Vector pGEX-4T-1-eIF5A

Objective To establish recombinant prokaryotic expression vector pGEX-4T-1-eIF5A and express the fusion protein. Methods Using pCMV6-XL4-eIF5A as template, the specific primers were designed. After PCR amplification, product was cloned into pGEX-4T-1 vector, the recombinant pGEX-4T-1-eIF5A was transferred to E.coli TOP10, then, it was identified with double restriction enzymes digestion analysis and DNA sequencing. The recombinant pGEX-4T-1-eIF5A which was identical with GeneBank was transformed into E.coli BL21. The expression of human eIF5A protein was analyzed by Western blot and SDS-PAGE assay with GST pull-down after IPTG induction. Results The construction of prokaryotic plasmid pGEX-4T-1-eIF5A was achieved. Human eIF5A sequence was identical with the GeneBank. E.coli BL21 expressed the glutathione S-transferase (GST)fusion protein of human eIF5A which was demonstrated by Western blot and SDS-PAGE assay with GST pull-down. Preliminary function study showed that eIF5A played a critical role in cell proliferation. Conclusion The construction and the expression of prokaryotic plasmid GST fusion protein of human eIF5A in E.coli BL21 was achieved successfully, and provide assistance for further study of the structure of the human eIF5A proteins and physiological functions such as the promotion of wound healing.