剪接因子SRp55原核表达质粒的构建和表达  

Expression of splicing factor SRp55 in E.coli cells

作　　者：尹晓敏[1] 施建华[1] 刘飞[1] 

机构地区：[1]南通大学江苏省神经再生重点实验室,南通226001

出　　处：《南通大学学报（医学版）》2007年第2期79-82,共4页Journal of Nantong University(Medical sciences)

基　　金：国家自然科学基金资助项目(30572076);江苏省自然科学基金资助项目(BK2004047);江苏省"六大人才高峰"资助项目

摘　　要：目的:本研究通过扩增剪接因子SR蛋白家族的SRp55基因,构建GST融合蛋白原核表达质粒pGEX-2T/SRp55,并在大肠杆菌中诱导表达并进行纯化。方法:用PCR法扩增SRp55基因,将扩增产物和载体pGEX-2T进行双酶切后回收,连接载体和目的片段,获得重组质粒。将重组质粒转化大肠杆菌BL21(DE3)。在大肠杆菌BL21(DE3)中通过IPTG进行诱导表达,然后用谷胱甘肽-琼脂糖球珠,亲和纯化得到纯的GST-SRp55融合蛋白。结果:SRp55基因以正确的阅读框架插入pGEX-2T。IPTG诱导大肠杆菌BL21(DE3)表达,随诱导时间的增加蛋白表达量增高,4h以后接近最高表达量。通过亲和纯化得到分子量在60kD左右的蛋白,经蛋白印迹分析证实为GST-SRp55融合蛋白。结论:成功地构建了GST融合蛋白原核表达质粒pGEX-2T/SRp55,并获得GST-SRp55融合蛋白,为进一步研究tau蛋白可变剪接机制提供了必要的基础。Objective： To express and purify Glutathione S Transferase（GST）-fusion protein of SRp55, a splicing factor, in E. coli cells. Methods： The eDNA of SRp55 was amplified by polymerase chain reaction （PCR） and inserted into pGEX-2T with BamH1 and EcoR1 restriction sites. The recombinant plasmid of pGEX-2T/SRp55 was transformed into E.coli cells to express GST-SRp55. The expressed GST-SRp55 in the E.coli cells was purified from the extract by affinity purification. The purity of GST-SRp55 was analyzed by SDS-PAGE or Western blot. Results： The SRp55 was inserted into pGEX-2T with correct open read frame. The expression of GST-SRp55 could be induced by adding Isopropyl 15-D-1-thiogalactopyranoside （IPTG） in a time-dependent manner. The expression level achieved the highest after adding IPTG for 4 hours. The purified GST-SRp55 by glutathione （GSH）-agarose beads showed a - 60 kD major band by coomassie blue staining, which also was recognized by anti-GST antibody. Conclusion： GST-SRp55 is expressed and purified successfully from E.coli cells ,which will help us to further study the regulation of tau splicing.

关 键 词：SRp55 TAU 原核表达 剪接因子 

分 类 号：Q786[生物学—分子生物学]