Abstract：The warm temperature acclimation related 65 kD protein-2 (Wap65-2) is a glycoprotein and so far only found in teleost fish. The expression pattern of Wap65-2 discloses its function in response to the stresses of temperature, infection and heavy metal contamination, and especially the function in anti-bacterial immunology attracts broad attention in recent years. In this study, the recombinant ayu (Plecoglossus altivelis) Wap65-2 mature peptide (rPaWap65-2m) was expressed in an insect baculovirus vector system (BEVS), by which we sought to explore a new way to produce biologically active rPaWap65-2m. The recombinant Bacmid-PaWap65-2 was obtained by cloning, and then used to transfect Spodoptera frugiperda cells (Sf9) for the viral stock. Sf9 cells in exponential growth phase were infected with high-titer P3 viral stock and then detected the expression of rPaWap65-2m by Western blot. Sf9 cells were cultured in optimized large scale serum-free system for rPaWap65-2m expression. rPaWap65-2m was purified by anion-exchange chromatography and nickel chelate affinity chromatography from the collected culture supernatant. The interaction between rPaWap65-2m and Complement 3 (C3) was identified by His-tag pulldown method. Results showed that after P3 viral stock infected Sf9 cells, rPaWap65-2m was expressed and secreted into the culture supernatant as a non-glycoprotein detected by Western blot. The maximum expression was obtained when Sf9 cells were infected with the multiplicity of infection (MOI) of 5 and collected at 72 h after infection. The purity of rPaWap65-2m was greater than 93%. rPaWap65-2m was verified to interact with full-length nature C3 by pulldown. In summary, the biologically active rPaWap65-2m was obtained for further study on its immunological function.
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