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Abstract Full Text

Expression of chicken (Gallus gallus) IL-4 in baculovirus vector system and its bioactivity analysis.

Abstract

Chicken (Gallus gallus) interleukin-4 (IL-4), which is a key indicator for Th2 type immune response in poultry, is very important in immune response and immune function analysis of chickens (Gallus gallus). Due to the lack of effective assays, the study of chicken IL-4 (ChIL-4) is still lagged behind other cytokines. To prepare recombinant ChIL-4 with high bioactivities, molecular biotechnology was used to construct recombinant vector pBac-ChIL-4, and then pBac-ChIL-4 expressed in Spodoptera furgiperda 9 (Sf9) cells. Firstly, ChIL-4 cDNA fragment was digested from plasmid pVAX1-ChIL-4 constructed previously with restriction enzymes and then subcloned to vector pFastBac1 to construct a new plasmid pFast-ChIL-4. With site-specific transposition, ChIL-4 cDNA fragment was transformed to vector bacmid in the baculovirus vector system to form recombinant plasmid pBac-ChIL-4. Secondly, pBac-ChIL-4 was transfected to Sf9 cells to generate recombinant ChIL-4 protein (rChIL-4 or Bac-ChIL-4). Finally, infected Sf9 cells were identified by immunofluorescent assay (IFA) for its expression of ChIL-4, and the supernatants of Sf9 cells infected were harvested and further analyzed by indirect enzyme-linked immuno sorbent assay (ELISA) and lymphocytes proliferation assay. The results of IFA indirect ELISA showed that rChIL-4 successfully expressed in the baculovirus vector system with bright green fluorescence observed in transfected Sf9 cells, polyclonal antibody against ChIL-4 generated previously could only reacted with rChIL-4 (Bac-ChIL-4, His-ChIL-4), but not other irrelative proteins (bovine IL-4, chicken IFN-γ) revealed by ELISA. Splenocytes proliferation assay demonstrated that rChIL-4 had good bioactivity in stimulating and activating T lymphocytes. This study not only facilitates the structural and functional analysis of ChIL-4, but also provides helpful reference value for the expression of other cytokines.