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当前位置: 首页 > 文章 > 敖汉细毛羊Notch1基因真核表达载体的构建及在成纤维细胞中表达量的研究 畜牧与兽医 2019 (3) 1-6
Position: Home > Articles > Construction of Notch1 plasmid in Aohan Fine Wool Sheep and expression of the gene in fibroblasts Animal Husbandry & Veterinary Medicine 2019 (3) 1-6

敖汉细毛羊Notch1基因真核表达载体的构建及在成纤维细胞中表达量的研究

作  者：
赵然然;张梦瑶;刘开东;王国义;刘积凤;柳楠;贺建宁
单  位：
青岛农业大学动物科技学院;青岛畜牧兽医研究所;赤峰市敖汉旗种羊场
关键词：
成纤维细胞;表达载体构建;Notch1转染;RT-PCR;基因表达
摘  要：
本研究旨在构建敖汉细毛羊Notch1基因表达载体及转染成纤维细胞后基因表达量变化。试验以敖汉细毛羊40日龄胎儿为研究对象,提取RNA反转录并参照Gen Bank中Notch1基因序列信息设计1对引物,通过PCR反应扩增获得Notch1基因片段,将得到的Notch1基因片段连接至pGM-T载体,构建pGM-T-Notch1克隆载体并转化大肠杆菌(E.coli) DH5α感受态细胞,提取质粒进行酶切鉴定后构建pcDNA3.1-Notch1重组表达载体,转化大肠杆菌(E.coli) DH5α感受态细胞。将敖汉细毛羊的成纤维细胞进行分离培养,并将构建的pcDNA3.1-Notch1瞬时转染成纤维细胞,利用荧光定量PCR技术检测Notch1基因在成纤维细胞中表达量的变化。结果显示:经酶切、测序鉴定质粒pcDNA3.1-Notch1构建成功,并且转染成纤维细胞后Notch1基因的表达量升高,且转染组的表达量极显著地高于对照组(P<0.01)。结论:成功构建了敖汉细毛羊Notch1基因的表达载体,并且成功转染成纤维细胞,基因表达量明显升高,结果可为进一步研究其功能奠定基础。
译  名：
Construction of Notch1 plasmid in Aohan Fine Wool Sheep and expression of the gene in fibroblasts
作  者：
ZHAO Ranran;ZHANG Mengyao;LIU Kaidong;WANG Guoyi;LIU Jifeng;LIU Nan;HE Jianning;College of Animal Science and Technology,Qingdao Agricultural University;Qingdao Institute of Animal Science and Veterinary Medicine;Aohan Banner Breeding Sheep Farm;
单  位：
ZHAO Ranran%ZHANG Mengyao%LIU Kaidong%WANG Guoyi%LIU Jifeng%LIU Nan%HE Jianning%College of Animal Science and Technology,Qingdao Agricultural University%Qingdao Institute of Animal Science and Veterinary Medicine%Aohan Banner Breeding Sheep Farm
关键词：
fibroblast;;plasmid construction;;Notch1 transfection;;RT-PCR;;gene expression
摘  要：
This study was aimed to identify the expression of the Notch1 plasmid and the expression of the fibroblast gene.Firstly,we extracted RNA reverse transcription and referred to the Notch1 gene sequence in Gen Bank to design a pair of primers,and amplified the Notch1 fragment using PCR.We then ligated the obtained Notch1 gene into the pGM-T vector to construct pGM-T-Notch1 recombinant plasmids and transformed them into the E.coli DH5α competent cells.We identified the plasmids by restriction enzyme digestion.We constructed the recombinant plasmid pcDNA3.1-Notch1 and transformed it into the E.coli DH5α competent cell.Next,we transferred the pcDNA3.1-Notch1 plasmids into fibroblasts,and used qRT-PCR to detect the expression level of Notch1.The results showed that we successfully constructed the pcDNA3.1-Notch1 plasmid and identified them by enzyme and sequencing.The expression level of the Notch1 gene in the fibroblasts was much higher than in the control group(P<0.01).In conclusion:we constructed the plasmids successfully and transfected them into the fibroblasts.The gene expression was significantly increased.These results would lay a foundation for the further research on the function of the gene.

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