Porcine TNF-α high level expression and preparation of its polyclonal antibody

【Objective】 To determine the specificity and reactivity of porcine tumor necrosisfactor α(TNF-α)polyclonal antibody,and the effects of CSFV infection on TNF-α secretion of PK-15 cells were investigated,to lay a foundation for revealing the pathogenic mechanism of classical swine fever virus(CSFV).【Method】 The TNF-α gene was amplified by RT-PCR using RNA template extracted from CSFV infected PK-15 cells,to construct prokaryotic expression vector pGEX-4 T-1-TNF-α and transform BL21 competent cells to induce the expression of fusion protein. Purified and concentrated SPF level Kunming mice were immunized with TNF-α polyclonal antibody. At the same time,eukaryotic expression vector pcDNA3.0-TNF-α was constructed and transfected into HEK-293 T cell and PK-15 cell to express TNF-α. Titer,reactivity and specificity of TNF-α polyclonal antibody was detected by Western blotting,ELISA andindirect immunofluorescence methods.【Result】 Prokaryotic expression vector pGEX-4 T-1-TNF-α transformed BL21 competent cells could express about 43 kD fusion protein induced by IPTG,and it was mainly expressed in the form of inclusion body.The eukaryotic expression vector pcDNA3.0-TNF-α transfected HEK-293 T cells could express 25 kD fusion protein,which was mainly expressed in cytoplasm and evenly distributed. The prepared TNF-α polyclonal antibody could react well with the fusion protein TNF-α expressed in HEK-293 T cells and the endogenous protein TNF-α in PK-15 cells,and had a good reaction specificity and the antibody titer was as high as 1:8000. CSFV could up-regulate the expression of TNF-α secreted by PK-15 cells. The expression trend of TNF-α and its downstream factor(TRAF1)was basically consistent,therewas certain correlation between them.【Conclusion】 The TNF-α protein antibody has the characteristics of high effective valence,good reactivity and strong specificity,it can be used to detect the overexpression of TNF-α level in eukaryotic cells after CSFV infection. TNF-α can stimulate TRAF1 production and participate in TRAF1-related signaling pathway to play its biological function. The expression of TNF-α and TRAF1 is similar after CSFV infected PK-15 cells.These results suggest that CSFV can stimulate TNF-α and TRAF1 signaling pathways,and leads to the inflammatory reaction.