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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。
The polypyrimidine tract binding protein-associated splicing factor (PSF) is a multifunctional DNA / RNA binding protein that inhibits the expression of proto-oncogenes and has tumor suppressor effects in humans and mice. Using the total RNA of human fibroblasts as a material, the PSF protein was amplified by RT-PCR using PSF-specific primers and cloned into the prokaryotic expression vector pET-28a (+) to construct the recombinant expression plasmid pET- 28-PSF, transformed into E. coli BL21 (DE3) and induced expression. SDS-PAGE and Western blot analysis showed that PSF was expressed in soluble form in E. coli. Gel blocking analysis showed that the prokaryotic expression of PSF fusion protein in vitro with mVL30-1 RNA binding, indicating that prokaryotic expression products of PSF is likely to have complete biological activity. Which laid the foundation for further study on the biological function of PSF protein.