摘要
本研究构建了猪TRIM56(sTRIM56)全长及其截短体的真核表达质粒,并检测了其融合蛋白的表达和定位。首先,根据sTRIM56基因序列及结构特点,设计扩增全长及4个截短体的引物,提取猪肺泡巨噬细胞3D4/21的总RNA,RT-PCR扩增sTRIM56全长及截短体基因,并将其克隆至pXJ41真核表达载体,构建全长及其截短体的真核表达质粒。其次,将sTRIM56全长及截短体真核表达质粒转染HEK-293T细胞,Western blotting检测其蛋白表达;转染猪肺泡巨噬细胞3D4/21,间接免疫荧光试验(IFA)检测其亚细胞定位。双酶切和测序结果显示,sTRIM56全长及截短体的真核表达质粒构建成功;sTRIM56蛋白约为82 kDa,与预期相符,各截短体蛋白条带也与预期相符;sTRIM56全长及4个截短体蛋白均主要定位于细胞质中。本研究结果可为后续深入研究sTRIM56蛋白及其不同结构域的抗病毒作用及分子机制奠定基础。
The eukaryotic expression plasmids of fulllength and truncated swine TRIM56(sTRIM56)were constructed,and the expression and localization of fusion proteins were confirmed.According to the se quence and structural characteristics of sTRIM56 gene,the primers of fulllength and four truncations were de signed.The total RNA of porcine alveolar macrophages 3D4/21 was extracted.The fulllength and truncated genes were amplified by RTPCR and cloned into pXJ41 eukaryotic expression vector to construct their expres sion plasmids.The fulllength and truncated eukaryotic expression plasmids of sTRIM56 were transfected into HEK293T cells,and the protein expression was detected by Western blotting.The plasmids were also transfected into porcine alveolar macrophages 3D4/21 and the subcellular localization was detected by IFA.The re sults of double enzyme digestion and sequencing showed that the fulllength and truncated eukaryotic expres sion plasmids of sTRIM56 were successfully constructed.Western blotting results showed that the protein of sTRIM56 was about 82 kDa,and the bands of truncated sTRIM56 proteins were consistent with the expected sizes.IFA results showed that the fulllength and four truncated proteins of sTRIM56 were mainly localized in the cytoplasm.This research laid a foundation for further study on the antiviral effect and molecular mechanism of sTRIM56 protein and its different domains.
作者
姚来娣
吴香菊
齐静
丛晓燕
李均同
贾杏林
杜以军
Yao Laidi;Wu Xiangju;Qi Jing;Cong Xiaoyan;Li Juntong;Jia Xinglin;Du Yijun(Veterinary Medicine College of Hunan Agricultural University,Changsha 410128,China;Institute of Animal Science and Veterinary Medicine,Shandong Academy of Agricultural Sciences/Shandong Key Laboratory of Animal Disease Control and Breeding/Key Laboratory of Livestock and Poultry Multiomics,Ministry of Agriculture and Rural Affairs,Jinan 250100,China)
出处
《山东农业科学》
北大核心
2023年第9期10-16,共7页
Shandong Agricultural Sciences
基金
山东省自然科学基金项目(ZR2021ZD08,ZR2020QC196,ZR2020KC005,ZR2021MC139)
国家自然科学基金项目(32102710)
国家重点研发计划项目(2021YFD1800300)
山东省农业科学院农业科技创新工程项目(CXGC2023A21,CXGC2023G03)。
关键词
猪TRIM56
截短体
真核表达质粒
表达
定位
Swine TRIM56
Truncations
Eukaryotic expression plasmids
Expression
Localization
