人PSF蛋白的原核表达及分析鉴定  

Prokaryotic Expression,Analysis and Identification of Human Polypyrimidine Tract Binding Protein-associated Splicing Factor

作　　者：王慧娟[1] 兰洋[1] 刘涛[1] 李灵[1,2] 

机构地区：[1]四川大学生命科学院功能基因组实验室,成都610064 [2]海南医学院海南省热带病重点实验室,海口571101

出　　处：《中国生物工程杂志》2011年第6期1-5,共5页China Biotechnology

基　　金：海南省自然科学基金(310045);国家自然科学基金(31000579)资助项目

摘　　要：PSF(polypyrimidine tract binding protein-associated splicing factor)蛋白是一种多功能的DNA/RNA结合蛋白,能够抑制原癌基因的表达,在人和小鼠中具有肿瘤抑制的作用。以人成纤维细胞总RNA为材料,利用PSF特异性引物通过RT-PCR的方法扩增得到PSF蛋白的编码序列,将其克隆到原核表达载体pET-28a(+)中构建重组表达质粒pET-28-PSF,转化大肠杆菌BL21(DE3)并诱导表达。SDS-PAGE及免疫印记分析表明PSF能够在大肠杆菌中以可溶性形式表达。凝胶阻滞分析显示,原核表达的PSF融合蛋白在体外条件下能够与mVL30-1 RNA结合,表明PSF的原核表达产物很可能具有完整的生物学活性。为进一步研究PSF蛋白的生物学功能奠定了基础。Polypyrimidine tract binding protein-associated splicing factor （PSF）, a muhifunctional RNA/ DNA-binding protein, can suppress the expression of proto-oncogene and function as tumor suppressor protein （TSP） in human and mouse. Using total RNA extracted from human fibroblasts, PSF-encoding cDNA was synthesized and inserted into vector pET-28a（ ＋ ） to construct the recombinant plasmid pET-28-PSF. The PSF fused with His-tag was expressed in E. coli BL21 （DE3） by IPTG induction and then purified by affinity chromatography. SDS-PAGE and Western blotting results indicated the soluble His-tagged PSF could be produced with a prokaryotic expression system. Finally, the RNA-binding capacity of recombinant PSF to mVL30-1 RNA was determined by Gel-shift assay. The results indicate that recombinant PSF produced with a prokaryotic expression system can be biologically active and will therefore allow us to investigate the function of PSF in the future.

关 键 词：PSF蛋白 肿瘤 原核表达 

分 类 号：Q786[生物学—分子生物学]