Prokaryotic expression and transmembrane transfer of fusion protein TAT-RIG-I-GFP.
- Author:
Xiaodan SHENG
1
;
Dihai HUANG
1
;
Hui GUO
1
;
Xia LIU
1
;
Zhuoming QIN
1
Author Information
- Publication Type:Journal Article
- Keywords: RIG-I; TAT; fusion protein; transmembrane delivery
- MeSH: Cell Membrane; DNA Primers; Escherichia coli; Gene Expression; Gene Products, tat; Genetic Vectors; Recombinant Fusion Proteins
- From: Chinese Journal of Biotechnology 2019;35(8):1463-1468
- CountryChina
- Language:Chinese
- Abstract: 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.
