hPSF蛋白的原核表达质粒的构建及分析鉴定

目的:构建hPSF蛋白原核表达质粒,在体外观察其与GAGE6调控区结合现象。方法:从人成纤维细胞中提取总RNA,利用hPSF特异性引物通过RT-PCR方法扩增hPSF cDNA编码序列,克隆入pET-28a载体中,构建重组载体pET-28-hPSF。将重组质粒转化入大肠埃希菌BL21(DE3)中进行表达,通过SDS-PAGE和免疫印迹法对表达产物进行鉴定。结果:酶切及测序结果显示,重组质粒构建成功;SDS-PAGE分析在相对分子质量约100kD处出现了1条蛋白条带,免疫印迹结果显示,所构建的重组载体可在大肠杆菌中高效表达hPSF蛋白。结论:成功构建了hPSF原核表达载体,并能够在大肠埃希菌中有效表达hPSF,为进一步研究hPSF的生物学作用奠定了基础。

Construction and identification of prokaryotic expression plasmid of human polypyrimidine tract binding protein-associated splicing factor

Objective: To construct the prokaryotic expression plasmid of human polypyrimidine tract binding protein-associated splicing factor(hPSF),and to explore the biological function and mechanism of hPSF.Methods: Total RNA was extracted from human fibroblasts,hPSF cDNA was amplified by RT-PCR using specific primers,then the cDNA was inserted into vector pET-28a to construct the recombinant vector pET-28-hPSF.The recombinant plasmid was transduced into Escherichia coli BL21(DE3) and the expression protein was detected by SDS-PAGE and Western bloting.Results:A specific fragment with 2 124 bp was amplified by PCR,and cloned into pET-28a(+).The homology of sequence was confirmed by comparing to GeneBank.The electrophoresis of recombinant plasmid pET-28-hPSF after enzyme incision displayed 2 DNA fragments with 5500 bp and 2 200 bp.The magnitude of expressed hPSF induced by IPTG was as expected.After Escherichia coli X2 Blue was transformed with recombinant vector pET-28-hPSF and induced with IPTG,then combinant protein with relative molecular mass about 100 kD was obtained.Conclusions: Recombinant expression vector pET-28-hPSF is constructed and expressed successfully,which will be helpful to our further research.