摘要
目的构建瓣状内切核酸酶1(Flap endonuclease 1,FEN1)基因重组真核表达质粒,并进行鉴定。方法提取L02细胞总RNA,逆转录合成cDNA,经巢式PCR扩增FEN1基因,定向克隆至载体pcDNA3.1,构建重组真核表达质粒,经酶切及测序进行鉴定。将鉴定正确的真核表达质粒转染293T细胞,Western blot检测FEN1蛋白的表达。结果 FEN1基因重组真核表达质粒经酶切及测序鉴定证明构建正确;在相对分子质量约47 000处可见目的蛋白条带,转染细胞中FEN1蛋白表达量较空载体转染组及空白细胞对照组提高约3倍。结论已成功构建了FEN1基因重组真核表达质粒,并可在293T细胞中过表达FEN1。
Objective To construct and identify the eukaryotic expression vector for flap endonuclease 1 （FEN1） gene. Methods Total RNA was extracted from L02 cells and reversely transcribed to cDNA, with which FEN1 gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3. 1. The constructed recombinant plasmid was identified by restriction analysis and sequencing, then transfected to 293T cells. The expressed FEN1 was identified by Western blot. Results Restriction analysis and sequencing proved that FEN1 gene was successfully inserted into plasmid pcDNA3. 1. The target protein band with a relative molecular mass of about 47 000 was observed on Western blot profile. As compared with those in 293T cells transfected with empty vector and the 293T cells untransfected, the expression level of FEN1 in 293T cells transfected with the constructed recombinant plasmid increased by about 3 folds. Conclusion The eukaryotic expression vector for FEN1 was successfully constructed, and FEN1 was over-expressed in 293T cells.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第2期181-183,187,共4页
Chinese Journal of Biologicals
基金
国家自然科学基金(81000732/H1904)资助项目
关键词
瓣状内切核酸酶1
真核细胞
基因表达
Flap endonuclease 1 （FEN1）
Eukaryotic cells
Gene expression
