猪Mx1基因及分段基因真核表达载体的构建和鉴定

Construction andidentification of porcine Mx1 and its segment genes

试验旨在通过对猪Mx1基因及其分段基因的真核表达载体的构建和鉴定,为进一步研究Mx1基因的功能奠定基础。试验以猪Mx1的基因序列为基础,以真核表达质粒pXJ41为载体,依据Mx1的蛋白结构进行分段,添加了Flag标签,构建了Mx1及Mx1分段基因的真核表达质粒,分别命名为pXJ41-Flag-Mx1、pXJ41-Flag-G、pXJ41-Flag-GMD、pXJ41-Flag-MDL4、pXJ41-Flag-GED。然后通过免疫印迹试验检测上述质粒在HEK-293T细胞中的表达情况,通过间接免疫荧光试验鉴定目的蛋白在细胞内的定位情况。免疫印迹试验成功检测到了Mx1及Mx1分段基因的蛋白条带,间接免疫荧光试验检测到各目的蛋白定位于细胞质。上述真核表达质粒可在HEK-293T细胞中成功表达,Mx1及其分段真核表达质粒的构建和成功表达及定位对进一步探索Mx1的功能及机制奠定了基础。

This study is aimed at constructing and identifying eukaryotic expression vectors of porcine Mx1 gene and its segment genes in order to lay a foundation for the in-depth study of the function of Mx1.Based on the gene sequence of the porcine Mx1 and taking pXJ41 as the vector,we divided Mx1 into several segments which were tagged with Flag and cloned into the eukaryotic expression plasmid pXJ41.The successful constructs were named pXJ-Flag-Mx1,pXJ41-Flag-G,pXJ-GMD,pXJ41-Flag-MDL4 and pXJ41-Flag-GED,respectively.The expression of these constructs in HEK-293T cells were identified by western blotting assay,and the location of the target proteins were identified by IFA.The protein bands of Mx1 and its segment genes were successfully detected by western blotting assay.IFA indicated that the target proteins were located in the cytoplasm.The eukaryotic expression vectors of the porcine Mx1 gene and its segment genes were successfully constructed and expressed in HEK-293T cells,which laid a foundation for further exploration of the function and antiviral mechanism of Mx1.