NDRG1干扰载体的构建及稳定过表达NDRG1人脑微血管内皮细胞的建立  

Construction of NDRG1 interference vector and establishment of human brain microvascular endothelial cells stably over-expressing NDRG1

作　　者：阮婷玉 史唯地 马勋[1] 王静[1] 康立超[2] 杜冬冬 殷月兰[3] RUAN Tingyu;SHI Weidi;MA Xun;WANG Jing;KANG Lichao;DU Dongdong;YIN Yuelan(Laboratory of Preventive Veterinary,College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;Analysis and Test Center,Xinjiang Academy of Agricultural Reclamation Sciences,Shihezi 832000,China;Key Laboratory of Zoonosis,Jiangsu Province,Yangzhou 225009,China)

机构地区：[1]石河子大学动物科技学院预防兽医实验室,新疆石河子832003 [2]新疆农垦科学院分析测试中心,新疆石河子832000 [3]江苏省人兽共患病学重点实验室,江苏扬州225009

出　　处：《吉林大学学报（医学版）》2022年第6期1614-1622,共9页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助项目(31860712,32160833,32160834);江苏省科技厅人兽共患病学重点实验室资助项目(R1901);新疆生产建设兵团科技局省部共建绵羊遗传改良与健康养殖国家重点实验室开放课题项目(MYSKLKF201905);新疆生产建设兵团动物疾病防控兵团重点实验室开放课题项目(2020BTDJ05)。

摘　　要：目的:构建N-myc下游调节基因1(NDRG1)的shRNA干扰载体和过表达载体,转染人脑微血管内皮细胞(hCMECs)后验证干扰和过表达效果并获得稳定过表达NDRG1的细胞。方法:将pGPU6-GFP质粒采用BamHⅠ和BbsⅠ双酶切后,分别与设计的8条shRNA连接构建pGPU6-GFP-NDRG1 shRNA干扰载体,经测序鉴定后采用Lipofectamine 2000转染hCMECs,采用荧光显微镜观察载体转染效率,将PCR扩增的NDRG1编码区序列与pMD19-T连接,经测序正确后将重组质粒与pcDNA3.1空质粒采用限制性内切酶BamHⅠ和HindⅢ双酶切,连接后构建pcDNA3.1-NDRG1过表达载体,采用Lipofectamine 2000将测序正确的过表达载体转染hCMECs,采用实时荧光定量PCR(RT-qPCR)法检测干扰和过表达的hCMECs中NDRG1 mRNA表达水平,Western blotting法检测hCMECs中NDRG1蛋白表达水平,采用新霉素筛选出阳性克隆hCMECs。结果:pGPU6-GFP载体经双酶切后,电泳结果显示完全线性化,测序结果显示shRNA均成功连接至pGPU6-GFP载体;荧光显微镜观察载体转染效率约为70%;RT-qPCR法检测,有3个shRNA干扰载体的干扰效果良好,转染后hCMECs中NDRG1 mRNA表达水平分别约为对照组的26%、21%和13%;Western blotting法检测到对应的特异性蛋白条带,重组质粒pcDNA3.1-NDRG1经双酶切后,电泳结果可见大小为1185和5428 bp的2条DNA条带,测序结果显示NDRG1基因成功插入,成功制备阳性克隆hCMECs。RT-qPCR法检测,阳性克隆hCMEC中NDRG1 mRNA表达水平较对照组升高,约为对照组的25.96倍;Western blotting法检测,hCMEC中NDRG1蛋白表达水平较对照组升高。结论:成功构建pGPU6-GFP-NDRG1 shRNA干扰载体并验证干扰效果;成功构建pcDNA3.1-NDRG1过表达载体和稳定过表达NDRG1的hCMECs。Objective:To construct the shRNA interference vector and over-expression vector of N-myc downstream regulatory gene 1(NDRG1)and transfect the human cerebral microvascular endothelial cells(hCMECs),and to verify the interference and over-expression effects and to obtain the cells stably overexpressing NDRG1. Methods:The pGPU6-GFP plasmid was double digested by Bam H Ⅰ and Bbs Ⅰ,and then ligated with eight designed shRNAs to construct the pGPU6-GFP-NDRG1 shRNA interference vectors respectively,and the hCMECs were transfected by Lipofectamine 2000 after sequencing and identification,and the transfection efficiency of the vectors was observed under fluorescence microscope;the sequence of NDRG1 coding region amplified by PCR was ligated with the pMD19-T,after correct sequencing,the recombinant plasmid and the empty plasmid pcDNA3. 1 were digested with restriction endonucleases Bam H Ⅰ and Hind Ⅲ,and the pcDNA3. 1-NDRG1 over-expression vector was constructed after ligation;the hCMECs were transfected with the correctly sequenced over-expression vector by using Lipofectamine 2000;the expression levels of NDRG1 mRNA in the hCMECs were detected by real-time fluorescence quantitative PCR(RT-qPCR) method,and the expression levels of NDRG1 protein in the hCMECs were detected by Western blotting method;the positive clone hCMECs were screened by neomycin. Results:After the pGPU6-GFP vector was doubly digested,the electrophoresis results showed that it was completely linear,and the sequencing results showed that the shRNAs were all successfully connected to the pGPU6-GFP vector;the transfection efficiency of vector was about 70 % observed under fluorescence microscope;the RT-qPCR results showed that there were three shRNA interference vectors with good interference effects, and the expression levels of NDRG1 mRNA in the hCMECs after transfection were about 26%,21%,and 13% of those in control group,respectively;the corresponding specific protein bands were obtained by Western blotting method,the recombinant plasmid pcDNA

关 键 词：N-myc下游调节基因1 RNA干扰 真核表达载体 过表达 人脑微血管内皮细胞 

分 类 号：R394.2[医药卫生—医学遗传学]