pAc5.1-luciferase-Kr-h1-3\'UTR荧光素酶报告基因载体的构建及鉴定
首发时间:2017-11-20
摘要:为研究miRNA对Kr-h1基因的调控,构建含有Kr-h1-3\'UTR基因序列的荧光素酶报告基因载体。实验利用PCR技术分别扩增获得luciferase基因和Kr-h1基因的3\'UTR序列,克隆入果蝇细胞表达载体pAc5.1/V5-HisB载体中。经双酶切和测序分析鉴定后,提取去内毒素质粒,转染入果蝇Kc细胞。利用荧光素酶检测系统检测表达情况。结果表明:pAc5.1-luciferase-Kr-h1-3\'UTR细胞表达载体构建成功并可在细胞中正常表达。
关键词: Kr-h1 荧光素酶报告基因 Kc细胞 miRNA
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Construction of the pAc5.1-luciferase-Kr-h1-3\'UTR luciferase reporter vector and verification
Abstract:To study the regulation of miRNA on Kr-h1, the luciferase reporter vector containing the 3\'UTR sequence of Kr-h1 gene was constructed The sequences encoding luciferase and 3\'UTR of Kr-h1 were amplified by PCR, and subcloned into pAc5.1/V5-HisB expression vector. After restriction enzymes digestion and DNA sequencing verification, the endotoxin removal plasmids were extracted, and transfected into Kc cells. The expression levels were detected by luciferase assay system. And the results showed that the pAc5.1-luciferase-Kr-h1-3\'UTR expression vector was constructed successfully and could normally expressed in Kc cells.
Keywords: Kr-h1 luciferase reporter Kc cell miRNA
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pAc5.1-luciferase-Kr-h1-3\'UTR荧光素酶报告基因载体的构建及鉴定
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