Study On The Pig C3d Molecular Adjuvant Enhancing The Effect Of PRRSV GP5 DNA Vaccine

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 leastcleavage which can't been digested by protease and can covalent linked to theantigen.It can attach to the CR2 which is on the surface of antigen-presentingcells(APC), so it cuts down the activation threshold of lymphocyte and elevates the immunity of the antigen.C3d has been defined to be an effcctivemolecular adjuvant.Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) was the pathogeny of Porcine Reproductive and Respiratory Syndrome (PRRS) represented by abortion of sew and respiratory obstacle of piglets. For its infectiousness as well as the immune repression and intercurrent brought to pigs,PRRSV caused huge economic loss to pig's feeding industry. Moreover,the mutants of PRRSV genes increase the difficulty to control the illness. Glycoprotein 5(GP5) is the primary structural protein of PRRSV. It shows the function of combining the virus to the receptor on the host cell as well as inducing the apoptosis of the cell and novel vaccine was designed according to its ability to induce the neutralization antibody. So GP5 provides the virus with important physiological functions and shows great value on the control of PRRS.The present study investigated the immunogenicity and protective efficacy of DNA vaccine encoding the GP5 gene fused to complement P28 (complement C3d receptor binding domain),and evaluated the immunogenicity to the fusion protein of GP5 with different copies of pig homolog complement C3d.The main results are as follows:After activating the liver tissue of pig 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 pig C3d cDNA,it's homology is 99.9%,it's proved that the C3d cDNA was successfully cloned. Compared the CR2 binding area with mammalian's. The sequence distance of pig mammalian is 80%,but 40%between birds. It reveals that the CR2 binding area was genus-specific.After cloning the C3d cDNA of pig used the liver as the source of mRNA,a pair of primers were designed to subclone the P28 gene to the pUC19 plasmid. Several tandems of P28 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 P28.n,then cloned the products to pCDNA3.1(+) plasmid. After this,the GP5 Gene of PRRSV was cloned through RT-PCR and inserted to the upstream of the P28.n which is in the pCDNA-P28.n,and the DNA Vaccines containing GP5 gene against PRRSV with C3d-P28 as molecular adjuvant were constructed. In addition,indirect immunofluorescence assay (IFA) could be found within the cells after Marc145 cells tranfected by recombinant plasmids. Furthermore,seven groups of mice were injected with these recombinant plasmids. GP5-specific ELISA antibody,IFN-γlevel and IL-4 level in the sera were detected. Serological analysis showed that BALB/c mice that vaccinated with DNA vaccine expressing fusions of pcDNA3.1-GP5-p28 (two, four or six repeats) elicited a higher level of humoral response compared with mice that vaccinated with DNA vaccine expressing only the pcDNA3.1 vector and pcDNA3.1-GP5 group (P <0.05). The increase in the immune response elicited by six copies of p28 was highest.