Study On The DNA Vaccine Containing VP1 Gene Against DHV With C3d As Molecular Adjuvant

Genetic immunization or DNA vaccination has initiated a new era of vaccine research. The technology involves the inoculation of exogenous antigen plasmid DNA into a living host to elicit an immune response to a protein encoded on the plasmid. The plasmid DNA is expressed persistently and excites the immune system of the living host. 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.DHV has three constructive proteinVP1 VP3 and VP0. VP1 encodes the main antigen site and type specific neutral site of DHV. Using the VP1 gene as immunogen to develop DNA vaccination of DHV is considered a basic strategy.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.The study developed the VP1-C3d-pcDNA3.1 DNA vaccine using C3d as molecular adjuvant. And the animal experiment investigated the immunogenicity and protective efficacy of the DNA vaccine. The main results are as follows:1. Cloning and expression of DHV VP1 geneThe diluted DHV was inoculate into allantoic cavity of SPF chick embryo. Total cell RNA were extracted from the allantoic fluid by liquid nitrogen -grinding method. The cDNA of VP1 was amplified by reverse transcription polymerase chain reaction (RT-PCR). In order to obtain recombinant env, an expression plasmid pGEX-4T-3-env was constructed and transformed into E.coli BL21strain. SDS-PAGE proved that the VP1 gene was highly expressed in E.coli. The Western blot analysis showed the protein had good reactionogenicity.2. Cloning and identification of duck C3d cDNAAfter activating the liver tissue of Shaoxing Ma duck 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 chicken and human C3d cDNA, it's homology is 87.6% 46.9% respectively. It reveals that the CR2 binding area was genus-specific.3. Construction of DNA Vaccines Containing VP1 gene against DHV with C3d as Molecular AdjuvantC3d was inserted into pcDNA3.1(+) to construct eukaryotic expression plasmid C3d-pcDNA3.1. Then the VP1 gene was cloned into C3d-pcDNA3.1 to finish the DHV DNA vaccine.The 8-week-old female mice were divided into three groups. Groupâ… was immunized with VP1-C3d-pcDNA3.1.Groupâ…¡was immunized with PBS. Groupâ…¢was immunized with commercial vaccine.Blood was collected from immunized after the fourth immunization. The antibody titer of each group was detected using indirect ELISA. The results proved the humoral immunity of duck immunized withVP1-C3d-pcDN A3.1 was very high.