Optimization and bioactivity verification of porcine recombinant visfatin with high expression and low endotoxin content using pig liver as template.

In order to obtain the porcine recombinant visfatin protein with high expression and low endotoxin content, the current study aims to express and verify the biological activity of the purified porcine recombinant visfatin protein. Firstly, four different expression strains were successfully constructed. Then they were simultaneously induced at 37°C for 4 hours and 16°C for 16 hours. The results showed that Visfatin-pET28a-Transetta was the best strain with high protein expression and purity at 16°C induction for 16 hours. After that, endotoxin was reduced from the recombinant visfatin until the residual endotoxin was less than one endotoxin units per milliliter (EU/mL). Finally, the purified porcine recombinant visfatin protein was incubated with RAW264.7 cells. The results of cell counting kit-8 (CCK-8) showed the survival rate of the cells first increased and then decreased with the increase in visfatin concentration. When the concentration of visfatin was 700ng/mL, the survival rate of the cells was the highest. Thereafter, control (PBS), Visfatin and Visfatin+PolymyxinB (Ploy.B) groups were incubated with the RAW264.7 cells for 6 hours. Real-time quantitative polymerase chain reaction (RT-qPCR) and Enzyme Linked Immuno-Sorbent Assay (ELISA) results showed that, as compared to the control group, the expressions of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 in Visfatin group were significantly increased (P<0.05). However, there was no significant difference between the Visfatin and Visfatin+Poly.B groups, indicating that porcine recombinant visfatin protein promoted the inflammatory activity of RAW264.7 cells while the residual endotoxin did not play a role, suggesting biological activity of porcine recombinant visfatin protein.