microRNA-133b过表达载体的构建及在E ca 109细胞的表达鉴定

摘要

Objective To construct miRNA lentivirus vectors (plKD-Ubc-eGFP-U6-shRNA)that contains Human Hsa-miR-133b gene and successfully transfect into esophageal cancer cell lines Eca109 to lay the foundation for investigating the role of miRNA-133b in Eca109 esophageal cancer cells.Methods Accord-ing to transcriptions of Human Hsa-miR-133b gene,designed the target of shRNA to arrange primer syn-thesis.Annealed single-stranded primer into the double-stranded oligo sequence,ligated into the linearized double digestion of RNA interference vector,replaced the original ccdB toxic gene of lentiviral vector, transferred the product into the competent cells and measured the sequence.Screened positive clones were lentivirus vector plasmid which interfered by target gene.Proceed the high purity plasmid extraction to the correctly verified and sequenced clones.Transfect the recombined plasmid into the Eca109 esophageal canc-er cell with the way of liposome.Results Successfully construct miRNA interfered lentivirus vectors,it was confirmed as recombined plasmids by PCR and that DNA oligo was successfully connected to the vec-tor and obtained Eca109 esophageal cancer cells which were stably transfected by recombined plasmid.The Eca109 cell transfected by recombined empty vector and recombined over-expressed vector were respectively observed with the different intensities of green fluorescence under inverted fluorescent micro-scope.Conclusion The experiment showed successfully construction of interfered lentiviral vector of the human miR-133b gene,and high expression was obtained after the transfection to esophageal cancer cells with liposome,which laid the foundation for further studying on the function of miR-133b.%目的：构建携带 Human Hsa-mir-133b 基因的 miRNA 干扰慢病毒载体（plKD-Ubc-eGFP-U6-shRNA），并成功转染食管癌 Eca109细胞株。为研究微小 RNA133b（miR-133b）对食管癌 Eca109细胞的作用奠定基础。方法凭据 Human Hsa-miR-133b基因的转录目录，设计 shRNA的靶点并进行引物的合成。将单链的引物退火成带粘性末端的双链片段，将其衔接于双酶切的 RNA干扰载体，替换原有的 ccdB毒性基因，用重组的质粒转染感受态细胞，并测定序列。筛选出来的阳性克隆即为 miR-133b的干扰载体质粒。提取测序结果准确的克隆里含有的载体质粒。将重组的载体质粒转染至食管癌 Eca109细胞。结果成功构建了 miR-133b干扰慢病毒载体，重组质粒进行PCR鉴定及序列测定证实DNA oligo成功连到载体中，并实现食管癌 ECA109细胞中重组载体质粒的稳定转染。在荧光倒置显微镜下分别观察到重组空载体组转染的 Eca109细胞和过表达载体组转染的Eca109细胞发出不同强度的带有绿色荧光的信号。结论该实验成功构建了人 miR-133b基因的干扰慢病毒载体，采取脂质体法对食管癌 Eca109细胞进行质粒转染，获得高表达，为进一步研究 miR-133b 的功能奠定牢固的基础。