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
en Zh
| WPRIM
| ID: wpr-771783
Biblioteca responsable:
WPRO
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.
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Asunto principal:
Proteínas Recombinantes de Fusión
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Expresión Génica
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Productos del Gen tat
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Membrana Celular
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Cartilla de ADN
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Escherichia coli
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Vectores Genéticos
Idioma:
Zh
Revista:
Chinese Journal of Biotechnology
Año:
2019
Tipo del documento:
Article