MAVS ΔTM基因真核表达载体的构建及其在293T细胞中的表达定位

Construction of Eukaryotic Expression Vector of MAVS ΔTM Gene and Expression Localization in 293T Cells

线粒体抗病毒信号蛋白(mitochondria antiviral signaling protein,MAVS)在脊椎动物的抗病毒免疫反应中起着非常重要的作用。为了后续研究MAVS跨膜区氨基酸在病毒侵染宿主细胞时所发挥的作用,通过基因工程方法,构建了MAVS基因C端跨膜结构域缺失突变体(MAVS ΔTM)。首先通过PCR技术扩增MAVS ΔTM基因片段,通过无缝克隆技术连接到pcDNA3.0-Flag载体上,后经酶切鉴定及测序结果表明MAVS ΔTM基因成功克隆到pcDNA3.0-Flag载体上,并将重组质粒定名pcDNA3.0-Flag-MAVS ΔTM。将重组质粒pcDNA3.0-Flag-MAVS ΔTM转染至人肾上皮细胞(HEK-293T)细胞中,利用蛋白免疫印迹实验和间接免疫荧光技术检测pcDNA3.0-Flag-MAVS ΔTM的表达以及定位情况。结果显示,pcDNA3.0-Flag-MAVS ΔTM在293T细胞中成功表达,并且均匀分布于细胞中。研究将为进一步研究MAVS蛋白跨膜区氨基酸的功能奠定基础。

Mitochondrial antiviral signaling proteins(mitochondria antiviral signaling protein,MAVS)played an important role in the antiviral immune response of vertebrates.In order to further study the role of amino acids in the transmembrane region of MAVS in virus infection of host cells,c-terminal transmembrane domain deletion mutants of MAVS gene(MAVS ΔTM)were constructed by genetic engineering method.Firstly,the MAVS ΔTM gene fragment was amplified by PCR technology and connected to the vector pcDNA3.0-Flag by seamless cloning technology.After enzyme digestion identification and sequencing results,it was confirmed that the MAVS ΔTM gene was successfully cloned into the vector pcDNA3.0-Flag,and the recombinant plasmid was named pcDNA3.0-Flag-MAVS ΔTM.The recombinant plasmid pcDNA3.0-Flag-MAVS ΔTM was transfected into human renal epithelial cells(HEK-293T),and the expression and localization of pcDNA3.0-Flag-MAVS ΔTM were detected by western blot and indirect immunofluorescence.The results showed that pcDNA3.0-Flag-MAVS ΔTM was successfully expressed in 293T cells and evenly distributed in cells,which would laid a foundation for further study on the function of amino acids in the transmembrane region of MAVS protein.