猪TRIM56全长及其截短体真核表达质粒的构建及蛋白的表达和定位

Construction of Swine TRIM56 Full-length and Its Truncated Eukaryotic Plasmids and Expression and Localization of Fusion Proteins

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.