中文摘要：

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I胛G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。

英文摘要：

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.