盐藻小G蛋白DsRab的原核表达及纯化  

Prokaryotic Expression and Purification of DsRab from Dunaliella salina

作　　者：李秀娟[1] 柴晓杰[1] 陶晓迎 赵欢[1] 丛玉婷[1] 

机构地区：[1]大连海洋大学农业部北方海水增养殖重点实验室辽宁省海洋生物资源恢复与生境修复重点实验室,大连116023

出　　处：《生物技术通报》2014年第4期176-180,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(30972240);辽宁省教育厅科技研究项目(2008T023)

摘　　要：前期对盐藻小G蛋白基因DsRab研究表明,在盐胁迫诱导下该基因转录水平明显提高。为进一步研究该蛋白在盐藻耐盐机制中的作用,PCR扩增DsRab的开放阅读框(ORF),并将其克隆至带有GST标签的原核表达载体pGS-21a,得到重组表达载体pGS-21a-DsRab。将重组表达载体转化E.coli BL21(DE3),IPTG诱导表达,并优化诱导表达条件,利用GST-SefinoseTM Kit进行纯化,用SDS-PAGE和Western blot鉴定。结果表明,成功构建了重组表达载体pGS-21a-DsRab,SDS-PAGE结果显示得到的蛋白与预期分子量相符,并且纯度较高;Western blot检测结果初步证明该融合蛋白为GST-DsRab。Previous studies indicated that the DsRab transcript could be increased by salt stress. In order to study the functions of DsRab in salinity tolerance, the open reading frame（ORF）of DsRab gene was obtained through PCR. The target fragment was cloned in pGS-21a, and the recombinant plasmid pGS-21a-DsRab was transformed into E. coli BL21（DE3）. The recombinant protein was induced with IPTG. Then the prokaryotic expression condition was optiminzed to harvested more supernatant recombinant protein. The products were purified by GST-SefinoseTM Kit, and identified by SDS-PAGE and Western blot. The results showed that the recombinant expression vector pGS-21a-DsRab was constructed successfully, the molecular weight of the recombinant protein was in the expected line. Western blot analysis showed that the recombinant protein can be identified specifically by the anti-GST antibody.

关 键 词：盐藻 DsRab GST 原核表达 纯化 

分 类 号：Q943.2[生物学—植物学]