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 h and 16 °C for 16 h. The results showed that Visfatin-pET28a-Transetta was the best strain with high protein expression and purity at 16 °C induction for 16 h. 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 700 ng/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 h. 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.

为了获得具有高表达和低内毒素含量的猪重组visfatin蛋白，本研究旨在表达和验证纯化的猪重组visfatin蛋白的生物学活性。首先，成功构建了四种不同的表达菌株。然后将它们同时在37°C诱导4 h和16°C诱导16 h。结果表明，Visfatin-pET28a-Transetta是在16°C诱导16 h时具有高蛋白表达和高纯度的最佳菌株。之后，将内毒素从重组visfatin中还原，直到残留的内毒素少于每毫升1个内毒素单位（EU / mL）。最后，将纯化的猪重组visfatin蛋白与RAW264.7细胞一起孵育。细胞计数试剂盒8（CCK-8）的结果显示，随着visfatin浓度的增加，细胞的存活率先增加然后降低。当visfatin浓度为700 ng / mL时，细胞的存活率最高。此后，将对照组（PBS），Visfatin和Visfatin +多粘菌素B（Ploy.B）组与RAW264.7细胞孵育6小时。实时定量聚合酶链反应（RT-qPCR）和酶联免疫吸附测定（ELISA）结果显示，与对照组相比，白介素（IL）-1β，肿瘤坏死因子（TNF）- Visfatin组的α和单核细胞趋化蛋白（MCP）-1显着增加（P <0.05）。但是，Visfatin和Visfatin + Poly.B组之间没有显着差异，