摘要
目的构建HSC70(HSC)真核表达质粒,并在HepG2细胞中表达。方法采用RT-RCR方法从正常人肝细胞系QZE中得到HSC70的cDNA序列,然后克隆入pcDNATM3.1/V5-HisA载体,构建重组质粒pcDNA-HSC70;经酶切和测序鉴定后,将pcDNA-HSC70体外转染HepG2细胞系,瞬时表达带有His标签的HSC70融合蛋白;并通过免疫细胞化学方法检测蛋白表达情况。结果RT-PCR扩增到1960bp的HSC70 cDNA片断,经测序分析与GenBank中已知序列一致;重组质粒pcDNA-HSC70转染后的HepG2细胞中,可检测到瞬时表达的HSC70-His融合蛋白。结论成功构建重组质粒pcDNA-HSC70,并可以在真核细胞HepG2中瞬时表达。
[Objective] To construct the HSC70 （heat shock cognate protein 70） eukaryotic expression plasmid and express it in HepG2 cells. [Methods] The HSC70 gene was amplified by RT-PCR from human QZE hepatocelhdar cells and cloned into the eukaryotic expression vector pcDNA^TM 3.1/V5-HisA, then the plasmid pcDNA-HSC70 was constructed. After identified by restriction endonuclease and sequencing,the plasmid was transfected into HepG2 cells to express HSC70-His fusion protein. The expression of HSC70-His was analyzed by immunocytochemical technique. [Results] 1960 bp fragment of HSC70 cDNA sequence was obtained by RT-PCR, and it was consistent with the known HSC70 sequence in GenBank. After the plasmid pcDNA-HSC70 being transfected into HepG2 cells, the fusion protein HSC70-His was instantaneously expressed in some of cells. [Conclusion] Recombinant eukaryotic expression plasmid pcDNA-HSC70 is successfully constructed and instantaneously expressed in HepG2 cells.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2008年第4期398-401,共4页
China Journal of Modern Medicine
基金
国家自然科学基金项目(编号:30170824)
关键词
HSC70
肝细胞
克隆
真核表达
heat shock cognate protein 70
hepatocyte
cloning
eukaryotic expression
