PL-JHAMT和PL-JHE荧光素酶报告基因载体的构建及鉴定
首发时间:2019-06-06
摘要:为研究miRNA对JHAMT和JHE基因的调控,构建分别含有JHAMT-3'UTR和JHE-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JHAMT和JHE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:PL-JHAMT和PL-JHE细胞表达载体构建成功并可在细胞中正常表达。
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Construction of the PL-JHAMT and PL-JHE luciferase reporter vector and verification
Abstract:To study the regulation of miRNA on JHAMT and JHE, the luciferase reporter vector containing the 3'UTR sequence of JHAMT and JHE gene were constructed. The sequences encoding luciferase and 3'UTR of JHAMT and JHE 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: luciferase reporter JHAMT JHE miRNA
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