Around the indicated days, chickens were euthanatized, lung samples were collected and subsequently homogenized and inoculated in SPF chicken embryos for the presence of infectious virus. immunize mice. In addition, Specific pathogen free (SPF) chickens were inoculated with the plasmids pcDNA-sM2, pcDNA-C3d-L1-C3d-L2-sM2, GST-sM2 and GST-C3d-L1-C3d-L2-sM2. The immune response was monitored by an enzyme-linked immunosorbent assay (ELISA) for sM2 antibody, and all the test animals were challenged with A/chicken/Bei Jing/WD9/98 (H9N2) virus. Results revealed that this anti-sM2 antibody in mice and chickens vaccinated with these proteins was higher than the nonfused forms of sM2, the GST-C3d-L1-C3d-L2-sM2 groups have conferred the highest 30% and 20% protection ratio in mice and chickens respectively. In addition, the pcDNA-C3d-L1-C3d-L2-sM2 also enhances the antibody responses to sM2 compared to pcDNA-sM2 in chickens, and acquired 13.3% protection ratio. Conclusion These results indicated that chicken C3d enhanced the humoral immunity against AIV M2 protein either fused proteins expressed by the prokaryotic system or with the DNA vaccine. Nevertheless, in view of the poor protection ratio for these animals, we speculated that this is not BMS-708163 (Avagacestat) a worthy developing of vaccine in these constructs. Background Complement is usually a protein system in the plasma of humans and animals . After being activated, a series of important biological reactions generate several complement proteins that nonspecifically defend against invading pathogens . While complement protein C3 is usually a central component of the innate immune system, it also plays an BMS-708163 (Avagacestat) important role in stimulating the humoral immune response [1,3]. At the point of convergence of three distinct pathways of complement activation, C3 is usually cleaved into C3a and C3b by the C3 convertase . Further proteolytic cleavage of C3b results in the formation of C3c and C3dg. The C3dg product can be further degraded by a variety of cellular proteases into C3d, a protein which attaches covalently to the surface of pathogens and upregulates B-cell responses [4,5]. Previous studies have exhibited that C3d could enhance antigen recognition and specific immunoglobulin synthesis by antigen-specific B cells, as the antigen is usually taken up and processed via cell receptor 2 (CR2) by both antigen-specific and non-specific B cells . Subsequent investigations showed that three copies of murine C3d could dramatically enhance antibody responses to specific antigen, being 100-fold more effective than incomplete Freund’s adjuvant [7,8]. Ross reported that C3d could enhance antibody responses directed toward a specific antigen encoded by a DNA vaccine . A DNA vaccine expressing a fusion of hemagglutinin (HA) from influenza virus or measles virus fused to three copies of the murine homologue of C3d (mC3d) achieved an early and efficient immune response in mice. Fusion to C3d has been shown to increase the immunogenicity of the capsular polysaccharide antigen of em Streptococcus pneumoniae /em . Using DNA vaccination, various forms of envelope (Env) proteins of the human immunodeficiency virus type 1 (HIV-1) fused at the carboxyl terminus with C3d of murine complement, generated high-titer, long-lasting, neutralizing antibodies in mice . In addition, the human homologue of C3d (hC3d) also enhanced anti-Env antibodies in rabbits when it was fused to sgp120 . Recently, Wang reported that this bovine homologue of C3d (boC3d) coupled to the E2 envelope protein of bovine viral diarrhea virus greatly enhanced immunogenicity in mice . Liu also reported that chicken C3d-P29 linked to the F gene of Newcastle disease virus (NDV) enhanced immunogenicity in chickens . Logan GJ found C3d (3)-fusion markedly increase antibody responses to the AAV-encoded model antigen (hen egg lysozyme) with greater than 50-fold enhancement in responses . Comparison of the human, mouse and bovine C3d sequences showed 84.1% amino BMS-708163 (Avagacestat) acid homology between hC3d and mC3d and 80.5% homology between hC3d and boC3d, they either showed the function of immune adjuvant in mammalian model. Information around the function of avian C3d is usually scarce. Importantly, there are structural differences in the mammalian CD253 and avian immune systems, particularly the role of the bursa as one of the central immune organs in avian species. Avian influenza (AI), caused by avian influenza virus (AIV), is usually a highly contagious disease of birds. Current AI vaccines induce antibodies against HA and neuraminidase (NA), two major surface glycoproteins expressed BMS-708163 (Avagacestat) around the virus particles. However, due to rapid antigenic variation of HA and NA, AI vaccine can not protect avian against the new avian influenza virus strains. A vaccine that is less sensitive to the antigenic evolution of the virus would be a major improvement..