We studied the construction of fusion protein TAT-RIG-I-GFP prokaryotic expression vector and verified the function of TAT in transmembrane delivery. First, four pairs of specific primers were designed, and the RIG-I gene of Mallard Duck (Anas platyrhynchos) was cloned. Then, the pET-TAT-RIG-I-GFP and pET-RIG-I-GFP prokaryotic expression vectors were constructed. Meanwhile, they were converted to E. coli BL21 (DE3), which were induced to be expressed after culture. After the purification of His-60 nickel affinity chromatography column and the identification of SDS-PAGE, the purified TAT-RIG-I-GFP and RIG-I-GFP proteins were incubated to DF-1 cells. Finally, fluorescence microscopy was used to observe whether the corresponding fluorescence was produced in DF-1 cells. The results showed that pET-TAT-RIG-I-GFP fusion with TAT showed obvious green fluorescence in DF-1 cells. However, the pET-RIG-I-GFP without TAT cannot display green fluorescence. This shows that TAT-fused protein have successfully delivered DF-1 cells and play a key role in transmembrane delivery. In conclusion, these results provide a solid material basis for further study of antiviral drugs in poultry.
本研究旨在探讨融合蛋白TAT-RIG-I-GFP 原核表达载体的构建并验证TAT 在跨膜递送中的作用。首先设计了4 对特异性引物， 克隆了绿头鸭Anas platyrhynchos RIG-I 基因， 构建了pET-TAT-RIG-I-GFP 和pET-RIG-I-GFP 原核表达载体；转化至感受态DE3 细胞，经IPTG 诱导表达，利用His60 镍亲和层析柱纯化，进行SDS-PAGE；然后，将纯化后的上述两种表达蛋白分别孵育DF-1 细胞；最后利用荧光显微镜观察是否在DF-1细胞产生相应的荧光。结果证实，携带有TAT 的pET-TAT-RIG-I-GFP 融合蛋白在DF-1 细胞中显示出明显的绿色荧光；而不具有TAT 的pET-RIG-I-GFP 蛋白却不能显示绿色荧光。这表明携带TAT 的融合蛋白已成功进入DF-1细胞，并在跨膜递送过程中发挥了关键作用。上述为进一步研制家禽的抗病毒药物奠定了基础。.
Keywords: RIG-I; TAT; fusion protein; transmembrane delivery.