敖汉细毛羊Hoxa7基因表达载体的构建及在成纤维细胞中的表达  

Construction of Hoxa7 gene expression vector and its expression in fibroblasts of Aohan fine wool sheep

作　　者：郑仰清 张梦瑶 刘鸿艳 徐晶 张廷荣[1] ZHENG Yangqing;ZHANG Mengyao;LIU Hongyan;XU Jing;ZHANG Tingrong(College of Animal Science and Technology,Qingdao Agricultural University,Qingdao 266109,China)

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109

出　　处：《黑龙江畜牧兽医》2020年第11期19-24,166,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：山东省现代农业产业技术体系项目(SDAIT-08-13)。

摘　　要：为了构建敖汉细毛羊Hoxa7基因表达载体以及研究表达载体转染成纤维细胞后基因表达量的变化,试验从40日龄敖汉细毛羊肩部皮肤提取RNA并反转录成cDNA,参照GenBank中Hoxa7基因的CDS区序列设计1对引物;将PCR扩增获取的Hoxa7基因CDS片段连接到pEASYTM-T1载体上,构建pEASYTM-T1-Hoxa7克隆载体,将克隆载体转化至大肠杆菌DH5α感受态细胞中;从pEASYTM-T1载体上切下Hoxa7基因片段,连接到pcDNA3.1表达载体上,再次转化至大肠杆菌DH5α感受态细胞中;将重组表达载体pcDNA3.1-Hoxa7瞬时转染到传代的成纤维细胞中,利用实时荧光定量PCR技术、Western-blot方法检测成纤维细胞中Hoxa7基因的mRNA和蛋白表达情况。结果表明:重组表达载体pcDNA3.1-Hoxa7构建成功;瞬时转染到成纤维细胞中后, Hoxa7基因的mRNA和蛋白表达量均极显著升高(P<0.01)。说明敖汉细毛羊Hoxa7基因在成纤维细胞中能够高效表达。In order to construct the Hoxa7 gene expression vector of Aohan Fine Wool Sheep and to study the changes of gene expression after transfection with expression vector into fibroblasts,RNA was extracted from the shoulder skin of 40-day-old fetus in Aohan Fine Wool Sheep and reverse transcribed into cDNA.One pair of primers was designed according to the CDS region of Hoxa7 gene in GenBank.The Hoxa7 gene CDS fragment obtained by PCR amplification was connected to the pEASYTM-T1 vector to construct the pEASYTM-T1-Hoxa7 cloning vector,and the clone vector was transformed into competent Escherichia coli DH5αcompetent cells.The Hoxa7 gene fragment was then cut from the pEASYTM-T1 vector,connected to the pcDNA3.1 expression vector,and then transferred to the competent Escherichia coli DH5αcompetent cells.The recombinant pcDNA3.1-Hoxa7 expression vector was transiently transfected into the passaged fibroblasts.Quantitative real-time PCR and Western-blot assays were used to detect the expression of Hoxa7 gene and protein in fibroblasts.The results showed that the recombinant pcDNA3.1-Hoxa7 expression vector was successfully constructed.The mRNA and protein expression levels of Hoxa7 gene were significantly increased after transient transfection into fibroblasts(P<0.01).This indicated that Hoxa7 gene of Aohan Fine Wool Sheep was highly expressed in fibroblasts.

关 键 词：Hoxa7基因 重组表达载体 成纤维细胞 实时荧光定量PCR WESTERN-BLOT 

分 类 号：S826.89[农业科学—畜牧学] Q786[农业科学—畜牧兽医]