MAVS全长及其截短体真核表达载体的构建和鉴定  

Construction and identificaition of eukaryotic expression vectors for MAVS full-length and its truncate

作　　者：玉妹 毕冬琳 郑天倚 李琼毅[1] YU Mei;BI Donglin;ZHENG Tianyi;LI Qiongyi(Faculty of Life Sciences and Engineering,Northwest Minzu University,Lanzhou 730030,China)

机构地区：[1]西北民族大学生命科学与工程学院,甘肃兰州730030

出　　处：《浙江农业学报》2025年第1期49-60,共12页Acta Agriculturae Zhejiangensis

基　　金：甘肃省自然科学基金(23JRRA720);西北民族大学实验室开放项目(SYSKF-2023102)。

摘　　要：建立线粒体抗病毒蛋白(mitochondrial antiviral-signaling protein,MAVS)全长及截短体的真核表达载体,并检测其在HEK293细胞中的表达,为后续探索MAVS在病毒逃逸机制研究中的作用提供重要的实验材料。首先,根据NCBI数据库中人源MAVS的全长序列,设计截短体引物,通过PCR从cDNA模板中扩增出MAVS全长及不同截短体的基因片段,并将其克隆到pcDNA3.1-3xflag载体中。随后,将构建好的载体转染到HEK-293细胞中,利用RT-PCR技术检测扩增产物大小,与目的大小进行对比,用Western blot技术检测MAVS蛋白在293细胞中的表达情况。结果表明,成功构建了全长MAVS(MAVS-FL)及其截短体MAVS-△C、MAVS-△CP、MAVS-PBD、MAVS-△Tm的真核表达载体,Western blot结果表明,构建的载体蛋白在HEK-293中能够有效表达。本研究成功构建了MAVS全长及其截短体的真核表达载体,并在HEK-293细胞中表达了这些蛋白,为后续研究MAVS蛋白的功能和抗病毒机制提供了实验材料。The eukaryotic expression vectors for the full-length and truncated forms of the mitochondrial antiviral-signaling protein(MAVS)was established and their expression in HEK293 cells was detected,which provides important experimental materials for subsequent investigations into the viral escape mechanism mediated by MAVS.Firstly,the full-length human MAVS sequence from the NCBI database was analyzed to design primers for generating truncated forms.Gene fragments corresponding to the full-length and various truncated forms of MAVS were amplified from cDNA templates using PCR and cloned into the pcDNA3.1-3xflag vector.Subsequently,the constructed vectors were transfected into HEK-293 cells.RT-PCR was employed to verify the size of the amplified products,which were compared with the target sizes,and Western blot analysis was used to detect the expression of MAVS proteins in the 293 cells.Eukaryotic expression vectors for full-length MAVS(MAVS-FL)and its truncates MAVS-△C,MAVS-△CP,MAVS-PBD,and MAVS-△Tm were successfully constructed.Western blot results confirmed that the proteins encoded by these vectors were effectively expressed in HEK-293 cells.In this study,we successfully constructed eukaryotic expression vectors for the full-length and truncated forms of MAVS and expressed these proteins in HEK-293 cells.This work provides important experimental materials for further study of the functions and antiviral mechanisms of MAVS proteins.

关 键 词：线粒体抗病毒蛋白 真核表达载体 截短体构建 蛋白表达 

分 类 号：Q7[生物学—分子生物学]