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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2011, Vol. 31 Issue (06): 1-5    
研究报告     
人PSF蛋白的原核表达及分析鉴定
王慧娟1, 兰洋1, 刘涛1, 李灵1,2
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
Prokaryotic Expression, Analysis and Identification of Human Polypyrimidine Tract Binding Protein-associated Splicing Factor
WANG Hui-juan1, LAN Yang1, LIU Tao1, LI Ling1,2
1. Functional Genome Laboratory, College of Life Science, Sichuan University, Chengdu 610064, China;
2. Hainan Provincial Key Laboratory of Tropical Medicine, Hainan Medical School, Haikou 571101, China
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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。

关键词: PSF蛋白肿瘤原核表达    
Abstract:

Polypyrimidine tract binding protein-associated splicing factor (PSF), a multifunctional 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.

Key words: PSF    Tumor    Prokaryotic expression
收稿日期: 2011-02-21 出版日期: 2011-06-28
ZTFLH:  Q786  
基金资助:

海南省自然科学基金 (310045)、国家自然科学基金(31000579)资助项目

通讯作者: 李灵     E-mail: leolibeike@gmail.com
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引用本文:

王慧娟, 兰洋, 刘涛, 李灵. 人PSF蛋白的原核表达及分析鉴定[J]. 中国生物工程杂志, 2011, 31(06): 1-5.

WANG Hui-juan, LAN Yang, LIU Tao, LI Ling. Prokaryotic Expression, Analysis and Identification of Human Polypyrimidine Tract Binding Protein-associated Splicing Factor. China Biotechnology, 2011, 31(06): 1-5.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I06/1

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