摘要
目的构建HCV5’NCR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HCV5’NCR片段,定向克隆至表达质粒pSEAP2-Control的SEAP基因上游,构建HCV5’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Huh-7。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78%,5μmol和10μmolASODN对pdNCRSEAP发光强度的抑制率分别为31.2%和48.6%,而对pSEAP2-Control的SEAP表达无显著抑制作用(P>0.05)。结论重组质粒pdNCRSEAP的SEAP表达受HCV5’NCR调控,为以HCV5’NCR为靶位点的药物筛选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 5＇noncoding region. Methods The fragment of HCV 5＇noncodingregion （5＇NCR） was amplified by polymerase chain reaction （PCR）, and was immediately cloned the upstreamof the SEAP gene of SEAP2-Control,an SEAP eukaryotic expression plasmid. With the liposome transfection technique, the resulting recombinant plasmid pdNCRSEAP was transfected into hepatocytes Hnh-7, 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 5μmol and 10 μmol, respectively. Results The light emission intensity of pdNCRSEAP expression was 78% that of pSEAP2-Control. The inhibition rates of pNCRSEAP luminescence intensity affected by ASODN of 5 μmol and 10μtmol were 31.2% and 48.6%, 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
《深圳中西医结合杂志》
2005年第4期216-219,共4页
Shenzhen Journal of Integrated Traditional Chinese and Western Medicine
基金
深圳市科技计划项目(JH 200507120860A)
