体外转录小鼠MAGE-A3 mRNA在NIH/3T3细胞中的表达  

Expression of in vitro transcribed MAGE-A3 mRNA in NIH/3T3 cells

作　　者：陈鹤丹 林煊 谢颖 陈尧 杨君仪 王赟 张世超[1,2] 胡祖权 曾柱 刘丽娜[1,2,4] Chen Hedan;Lin Xucn;Xie Ying(Dept of Biotechnology,School of Biology and Enginering,Guizhog Meeical Unwersita,Guiyang 550025;Key Laboratory of Biology and Medical Engineering/Immune Celli and AntiCody Engineering ResearcC Center sf Guizhou Province/Enginering ResearcC Centes of Medical Biotechnology,Guizhog Meeical Unwersita,Guiyang 550025)

机构地区：[1]贵州医科大学生物与工程学院生物技术教研室,贵阳550025 [2]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,贵阳550025 [3]贵州医科大学基础医学院,贵阳550025 [4]贵州医科大学环境污染与疾病监控教育部重点实验室,贵阳550025

出　　处：《安徽医科大学学报》2020年第8期1157-1160,共4页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81760556);贵州省科技计划项目(编号:黔科合基础[2016]1118);贵州省科技合作计划项目(编号:黔科合LH字[2015]7318);贵州医科大学2018年度学术新苗培养及创新探索专项项目(编号:黔科合平台人才[2018]5779-55);贵阳市科技计划项目(编号:筑科合同[20161001]40、筑科合同[2017]5-27号);贵州省卫生健康委科学技术基金项目(编号:gzwjkj2019-1-041);国家级大学生创新创业训练计划项目(编号:20195200134)。

摘　　要：目的构建编码MAGE-A3的真核表达载体,体外转录获得MAGE-A3 mRNA,在哺乳动物细胞NIH/3T3中表达MAGE-A3蛋白。方法通过逆转录PCR从三阴型乳腺癌细胞系4T1中扩增得到MAGE-A3编码基因,插入到载体pcDNA3.1(+)中,成功构建重组表达载体pcDNA3.1-MAGE作为体外转录RNA的模板。利用T7体外转录试剂盒获得MAGE-A3 RNA,添加poly(A)尾后进行mRNA纯化。将纯化后的MAGE-A 3 mRNA转染到NIH/3T3细胞中,Western blot实验检测MAGE-A3蛋白的表达。结果pcDNA3.1-MAGE-A3正确表达和构建。体外转录MAGE-A3 mRNAA(260)/A(280)为1.92,浓度是872.3 ng/μl,长度约为500 bp。转染MAGE-A3 mRNA后,NIH/3T3能表达大小约35 ku的MAGE-A3蛋白。结论成功构建编码MAGE-A3的真核表达载体,体外转录获得纯度较高的MAGE-A3 mRNA,该mRNA能在NIH/3T3细胞中表达MAGE-A3蛋白。Objective To construct eukaryotic expression plasmid encoding MAGE-A3,prepare in vitro transcribed mRNA and express MAGE-A3 protein in NIH/3 T3 cells.Methods The whole gene of MAGE-A3 was amplified by RT-PCR and cloned into pcDNA3.1(+)expression vector to build pcDNA3.1-MAGE.Subsequently,the template DNA for transcription in vitro was prepared by PCR.Then,the template DNA was transcribed into MAGE-A3 RNAs using MEGAscript T7 transcription kit.The transcripts were added a poly(A)tail to the 3’termini and purified by using transcription clean-up kit.After transfection of the purified MAGE-A3 mRNA into NIH/3 T3 cells,Western blot analysis was used to confirm the expression of MAGE-A3 protein.Results The gene inserted into the recombinant vector was proven to be completely identical with the sequence of the MAGE-A3 gene in online database.The ratio of A260/A280 of the purified mRNA was 1.92,and the concentration was 872.3 ng/μl.The length of MAGE-A3 mRNA was about 500 bp.After MAGE-A3 mRNA transfected into NIH/3 T3 cells,MAGE-A3 protein could be produced in cells and the size of protein was 35 ku.Conclusion Eukaryotic expression plasmid encoding MAGE-A3 is successfully constructed and the high-purity MAGE-A3 mRNA is obtained in vitro.The mRNA can be translated into MAGE-A3 protein in NIH/3 T3 cells.

关 键 词：MAGE-A3 体外转录mRNA 蛋白表达 

分 类 号：Q812[生物学—生物工程]