Abstract:

【Objective】DNA vaccines containing PRRSV GP5 gene with porcine C3d-p28 were constructed and the enhancing effect of molecular adjuvant C3d-p28 was proved. 【Method】Pigs were injected with the oil emulsion of Escherichia coli inactivated vaccine in order to induce immune activation. After cloning the C3d cDNA of pig by using liver as the source of mRNA, a pair of primers was designed to sub clone the P28 gene to the pUC19 plasmid. Several tandems of p28 were constructed in the pUC19 plasmid using a pair of isoschizomers BamHI and Bgl II. The genes of p28.n, which were digested from pUC19-p28.n, were cloned into pcDNA3.1 (+) plasmid. The GP5 gene of PRRSV was cloned by RT-PCR and inserted into the upstream of pcDNA3.1-p28.n plasmid, and the DNA vaccines containing GP5 gene with C3d-p28 as molecular adjuvant were successfully constructed. All recombinant plasmids were verified by PCR, restriction endonuclease digestion and DNA sequencing. In addition, indirect immunofluorescence assay (IFA) could be found within the cells after Mark145 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. 【Result】It was showed that the C3d cDNA of pig was cloned and several tandems of P28 and pcDNA3.1 (+)-GP5-C3d-p28.n were successfully constructed. 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 the highest. 【Conclusion】The DNA vaccine containing PRRSV GP5 gene with porcine C3d-p28 has been successfully constructed, which could elicit humoral and cellular immune responses. The complement C3d–p28 can significantly enhance the nucleic acid vaccine immune effect as molecular adjuvant.