摘要
目的 :构建HCVIRES调控外分泌性碱性磷酸酶 (SEAP)基因表达的细胞模型。方法 :用PCR技术扩增HCV 5′NCR片段 ,定向克隆至表达质粒 pSEAP2 Control的SEAP基因上游 ,构建HCV 5′NCR调控SEAP表达的重组质粒 pdNCRSEAP。用脂质体基因转染技术 ,将pdNCRSEAP转染至肝细胞株QSG770 1。用化学发光法检测SEAP的表达 ,并观察不同浓度反义寡聚核苷酸 (ASODN)对SEAP表达的影响。结果 :重组质粒 pdNCRSEAP表达的发光强度为pSEAP2 Control的 76 %,5 μmol和 10 μmolASODN对pdNCRSEAP发光强度的抑制率分别为2 9.2 %和 44 .6 %,而对 pSEAP2 Control的SEAP表达无显著抑制作用 (P >0 .0 5 )结论 :重组质粒 pdNCRSEAP的SEAP表达受HCV 5′NCR调控 ,为以HCV 5′NCR为靶位点的药物评价建立了检测方便的细胞模型。
Objective To establish a cell model of secreted alkaline phosphatase (SEAP) controlled by HCV internal ribosome entry site ( IRES ). Methods The fragment of HCV 5′ noncoding region (5′ NCR ) was amplified by polymerase chain reaction ( PCR ), and was immediately cloned the upstream of the SEAP gene of pSEAP2-Control, an SEAP eukaryotic expression plasmid. With the liposome transfection technique, the resulting recombinant plasmid pdNCRSEAP was transfected into hepatocytes QSG7710, and the SEAP activity of cell culture media was monitored quantitatively by the chemiluminescent method. The regulatory effect of the HCV 5′ NCR on the SEAP expression was measured by the treatment of transfected cells with antisense oligodeoxynucleotide (ASODN) at 5 μmol and 10 μmol, respectively. Results The light emission intensity of pdNCRSEAP expression was 76% that of pSEAP2-Control. The inhibition rates of pNCRSEAP luminescence intensity affected by ASODN of 5 μmol and 10 μmol were 29.2% and 44.6%, respectively, while ASODN had no significant effect on the pSEAP2-Control expression. Conclusion The SEAP expression of pdNCRSEAP is controlled by HCV 5′NCR. The cell model of drug evaluation targeted at HCV 5′NCR is successfully established and can be analyzed conveniently.
出处
《湖南医科大学学报》
CSCD
北大核心
2003年第4期335-337,共3页
Bulletin of Hunan Medical University
基金
省医药卫生科技项目 (2 0 0 0 0 0 1)
湖南省科技攻关项目 (0 2ssy30 83)
关键词
HCV
IRES
调控
外分泌性碱性磷酸酶基因
肝细胞
表达
hepatitis C virus
internal ribosome entry site(IRES)
secreted alkaline phosphatase (SEAP)
QSG7701 cells
