【摘要】 背景:Claudin-1是一种多功能蛋白,除了组成紧密连接封闭细胞间隙,它在转录水平的表达失调可能作为一种分子标志反映到恶性肿瘤的侵袭转移和预后中。目的:构建重组大鼠重组Claudin-1萤光素酶报告基因质粒。方法:设计和合成包含5’非转录区的约2000bp的脱氧核糖核酸链,经过限制性内切酶KpnⅠ和MluⅠ酶切后插入到pGL3-Basic载体中,并用感受态E.coliDH5α和Pmd18-T-simple载体筛选阳性样品。阳性克隆通过测序和PCR证实。实验分为4组:对照组,阳性对照组(pGL3-promoter质粒转染),阴性对照组(转染pGL3-Basic质粒),实验组(转染pGL3-Claudin-1promoter质粒)。将质粒转染293T细胞检测Claudin-1启动子活性。结果与结论:重组质粒DNA测序结果采用NCBIblast比对与GenBank(gi|62750811)中大鼠Claudin-1基因启动子序列完全匹配。重组萤光素酶报告基因转录活性检测与pGL3-Basic质粒相比,重组pGL3质粒转录活性明显增强(P<0.001)。经过测序和转染结果证实有效的pGL3-Claudin-1启动子质粒构建成功。更多还原

【Abstract】 BACKGROUND:Claudin-1 is a multi-functional protein;besides,construction of tight junction strand to seal paracellular space,which expression disorder in transcriptional level may be also involved in malignant cancer for invasion,metastasis and prognosis as a molecular marker.OBJECTIVE:To construct recombinant rat Claudin-1 luciferase reporter plasmid.METHODS:Oligonucleotide containing about 2 000 bp in 5’-UTR of rat Claudin-1 genome DNA was designed and synthesized,which was inserted into pGL3-Basic vector after double digestion by restriction enzyme KpnⅠ and MluⅠ;competent E.coli DH5α and pMD18-T-simple vector were used for screening of positive sample.Positive clones were identified by PCR and sequencing.There were four groups in the experiment:Control group,positive control group(transfected by pGL3-promoter plasmid),negative group(transfected by pGL3-Basic plasmid) and experimental group(transfected by pGL3-Claudin-1 promoter plasmid).Claudin-1 promoter activity was detected in the 293T cell transiently transfected.RESULTS AND CONCLUSION:The result of recombinant pGL3 plasmid from DNA sequencing was fully consistent with promoter sequence of rat Claudin-1 gene in GenBank(gi|62750811) by NCBI blast assay.Compared with pGL3-basic plasmid,the transcriptional activity of recombinant luciferase report gene in recombinant pGL3 plasmid was obviously increased(P < 0.001).Gene sequencing and transfection results confirmed that effective pGL3-Claudin-1 promoter plasmid had been constructed successfully.更多还原