小鼠黏蛋白1(MUC1)基因的体外转录及其在NIH/3T3细胞中的表达  

Preparation of in vitro transcribed mouse MUC1 mRNA and its expression in NIH/3T3 cells

作　　者：林煊 陈鹤丹 谢颖 曾柱[1,4] 胡祖权 贾义[1,3] 王赟 谭承建[5] 刘丽娜 LIN Xuan;CHEN Hedan;XIE Ying;ZENG Zhu;HU Zuquan;JIA Yi;WANG Yun;TAN Chengjian;LIU Lina(Key Laboratory of Biology and Medical Engineering,Immune Cells and Antibody Engineering Research Center of Guizhou Province, Engineering Research Center of Medical Biotechnology,Guizhou Medical University,Guiyang 550025;State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550025;School of Biology and Engineering,Guizhou Medical University,Guiyang 550025;School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025;School of Ethnic Medicine,Guizhou Minzu University,Guiyang 550025,China)

机构地区：[1]贵州医科大学生物与医学工程重点实验室,贵州省免疫细胞与抗体工程研究中心,医药生物技术工程研究中心,贵州贵阳550025 [2]贵州医科大学省部共建药用植物功效与利用国家重点实验室,贵州贵阳550025 [3]贵州医科大学生物与工程学院,贵州贵阳550025 [4]贵州医科大学基础医学院,贵州贵阳550025 [5]贵州民族大学民族医药学院,贵州贵阳550025

出　　处：《细胞与分子免疫学杂志》2018年第10期896-901,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81760556,31660103);贵州省细胞与基因工程创新群体(黔教合KY字[2016]031);贵州省科学技术基金(黔科合基础[2016]1118);贵州省科技合作计划(黔科合LH字[2015]7318);贵阳市科技计划项目(筑科合同[20161001],筑科合同[2017]5-27号).

摘　　要：目的体外转录获得小鼠黏蛋白1(MUC1) mRNA并在NIH/3T3细胞中表达。方法通过反转录PCR从小鼠4T1细胞中扩增MUC1基因,然后插入真核表达载体pc DNA3. 1(+)中构建重组表达载体pc DNA3. 1(+)-MUC1。双酶切鉴定正确后,以该质粒为模板,下游引物中引入血凝素标签(HA TAG)序列,PCR扩增MUC1-HA TAG融合基因片段,然后克隆至表达载体pc DNA3. 1(+),重组载体经双酶切鉴定和DNA序列测定后作为模板,PCR扩增获得含T7启动子和MUC1-HA TAG融合基因片段的PCR产物作为体外转录模板,通过体外转录、加尾、纯化最终获得修饰的MUC1 mRNA。通过多种转染试剂将体外转录MUC1-HA TAG mRNA转入NIH/3T3细胞,Western blot法检测融合蛋白MUC1-HA TAG的表达。结果反转录PCR扩增的MUC1基因长度约为1900 bp,酶切鉴定和序列分析证实MUC1-HA TAG融合基因已成功插入pc DNA3. 1(+)质粒。插入序列与Gen Bank中小鼠的MUC1基因序列完全一致,HA TAG正确插入MUC1下游,读框正确。Western blot法分析证实体外转录修饰的MUC1-HA TAG mRNA能在NIH/3T3细胞中表达。结论体外转录修饰的小鼠MUC1-HA TAG mRNA能在哺乳动物NIH/3T3细胞中翻译表达。Objective To obtain in vitro transcription of mouse mucin 1( MUC1) mRNA and express it in NIH/3 T3 cells.Methods The MUC1 gene was amplified from mouse 4 T1 cells by reverse transcription PCR( RT-PCR),and then insertedinto the eukaryotic expression vector pc DNA3. 1( +) to construct the recombinant vector pc DNA3. 1( +)-MUC1. After identificationby restriction enzyme cutting,the recombinant plasmid was used as a PCR template for the amplification of the MUC1-HATAG fusion gene fragment with the reverse primer containing hemagglutinin tag( HA TAG) sequence. Subsequently,theMUC1-HA TAG fusion gene was cloned into the expression vector pc DNA3. 1( +). After double restriction enzyme digestionand DNA sequence identification,the recombinant plasmid encoding MUC1-HA TAG was used as a PCR template for theamplification of a PCR product containing T7 promoter and the MUC1-HA TAG fusion gene fragment as an in vitro transcriptiontemplate to obtain the modified MUC1-HA TAG mRNA by in vitro transcription,tailing and purification. The resultingMUC1-HA TAG mRNA was transfected into NIH/3 T3 cells by various transfection reagents and the expression of the fusionprotein MUC1-HA TAG was detected by Western blot analysis. Results The MUC1 gene amplified by RT-PCR was about1 900 bp in length. The restriction enzyme digestion and sequence analysis confirmed that the MUC1-HA TAG fusion genehad been successfully inserted into pc DNA3. 1( +),and the insertion sequence was identical to the MUC1 gene sequence ofthe mouse in GenBank database. HA TAG was correctly inserted in the downstream of MUC1 gene and the reading framewas correct. Western blot analysis indicated that the in vitro transcriptionally modified MUC1-HA TAG mRNA could beexpressed in NIH/3 T3 cells. Conclusion In vitro transcriptionally modified mouse MUC1-HA TAG mRNA can be translatedand expressed in mammalian NIH/3 T3 cells.

关 键 词：黏蛋白1(MUC1) 体外转录 蛋白表达 

分 类 号：Q812[生物学—生物工程] Q344.13