Prokaryotic expression and transmembrane transfer of fusion protein TAT-RIG-I-GFP / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 1463-1468, 2019.
Article
in Chinese
| WPRIM
| ID: wpr-771783
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Recombinant Fusion Proteins
/
Gene Expression
/
Gene Products, tat
/
Cell Membrane
/
DNA Primers
/
Escherichia coli
/
Genetic Vectors
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2019
Type:
Article
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