Study On The Adjuvant Effect Of Avaian C3d-P29 Gene

Genetic immunization or DNA vaccination has initiated a new era of vaccine research. The technology involves the inoculation of plasmid DNA into a living host to elicit an immune response to a protein encoded on the plasmid. The potential advantages of DNA vaccines include the induction of cellular and humoral immune responses,flexible genetic design, lack of infection risk, stability of reagents, and the relatively low cost of production in amicrobial host. .However the gene vaccine could not provocate high titer and long-lasting immune response.Several approaches were explored to enhance the immunity of DNA vaccines such as using molecular adjuvant.Complement C3 is the core factor of the classical pathway, alternative pathway and lectin pathway. Cleavages of C3 have extensive immune function. C3d is the final cleavage product of C3.It's been thought to be the least cleavage which can't been digested by protease and can covalent linked to the antigen.It can attach to the CR2 which is on the surface of antigen-presenting cells (APC),so it cuts down the activation threshold of lymphocyte and elevates the immunity of the antigen. C3d has been defined to be an effcctive molecular adjuvant.Recently DNA vaccines to ND have been actively studied. Most of them focus on the major glycoproteins of F and HN, which are involved in essential steps of viral infection. Glycoproteins of F also designed as fusion protein constitute the major virulence of ND,it plays a important role in the process of membrane fusion of virus peplos and host cell membrane ,it helps the duplication and taking off the nucleocapsid of the virus after penetrating into the host cell cytolymph. So F gene is the best choice to the gene vaccine against ND.The present study investigated the immunogenicity and protective efficacy of DNA vaccine encoding the F gene fused to complement P29 (complement C3d receptor binding domain), and evaluated the immunogenicity to the fusion protein of F with different copies of AAchicken homolog complement C3d. The main results are as follows:1. Cloning and identification of AA chickens C3d cDNAAfter activating the liver tissue of AA Chickens by Escherichia coli, extracted total cell RNA from the liver tissue by liquid nitrogen-grinding method. The cDNA of C3d was amplified by reverse transcription polymerase chain reaction (RT-PCR). The cDNA fragments were directly inserted into pMD18-T plasmid. Compared the result with Lai Hang chicken C3d cDNA, it's homology is 99.5%,it's proved that the C3d cDNA was successfully cloned. Compared the CR2 binding area with mammalian's. The sequence distance of chicken mammalian is less than 40%,but 80%between mammalian listed. It reveals that the CR2 binding area was genus-specific.2. Construction of DNA Vaccines Containing F gene against Newcastle Disease Virus with C3d-P29 as Molecular AdjuvantC3d is the final cleavage product of C3 and fusion to C3d enhances the immunogenicity of the antigen. CR2/CD21 plays a very important role in it's function.P29 is the gene encoding the binding area to CR2 of C3d.After cloning the C3d cDNA of AA broilers used the liver as the source of mRNA ,a pair of primers were designed to subclone the P29 gene to the pUC19 plasmid. Several tandems of P29 were constructed in the pUC19 plasmid used a pair of isoschizomers-BamH I and Bgl II. Digested the pUC- P29.n to get the gene of P29.n,then cloned the products to pCDNA3.1 (+) plasmid. After this, the F Gene of Newcastle disease virus (NDV ) was cloned through RT-PCR and inserted to the upstream of the P29.n which is in the pCDNA-P29.n, and the DNA Vaccines containing F gene against NDV with C3d-P29 as molecular adjuvant were constructed. Several groups of SPF chickens were injected with these recombinant plasmids. It is found that the pCDNA-F-P29.4 and pCDNA-F-P29.6 group has higher HI antibody titers than the pCDNA-F group. The pCDNA-F-P29.4 and pCDNA-F-P29.6 group's HI antibody titers doesn't achieve titers as high as the inactive vaccine group, however, they all can provide protection against the lethal F48E9 virus challenge. To date, published research into the adjuvant activities of C3d has been limited to experiments in mice, using antigens unrelated to diseases occurring naturally in these species and there is little information about chicken C3d. This research forms the basis for ongoing investigation of the chicken C3d.