Construction Of Recombinant Porcine Circovirus Type 2/Interleukin-2 Nucleic Acid Accine And Studies On Immune Effect

Porcine circovirus type 2 (Porcine circovirus type 2, PCV2) are the main pathogens and trigger Postweaning multisystemic wasting syndrome.ORF1 and ORF2 are main open reading frames in PCV2,the ORF2 gene is an important antigen genes, the protein-coding (Cap protein), the existence of epitope in Cap epitope, with immunogenicity, so ORF2 gene is an objective gene which use establishing PCV2 specific serology and researching DNA vaccine of the PCV2. Current, no available PCV2 vaccine in domestic and foreign, the development of PCV2 DNA vaccine to control disease of PCV2 is of great significance.The primers with double restriction site were designed and amplify ORF2 gene of PCV2.The ORF2 gene was cloned into pGEM-T easy cloning vector. The recombinant chimeric gene of PCV2-linker-PoIL-2 was constructed by PCV2 ORF2 gene linked porcine interleukin-2 (PoIL-2) mature peptide gene via a 15-amino acid glycine-rich linker [linker(,G4S)3] by SOE-PCR (splicing by overlap extension-PCR). The recombinant chimeric gene was cloned into pGEM-T Easy vector and subsequently into eukaryotic expression pcDNA3.1(+) vector. The positive recombinant expression plasmid rpcDNA3.1/PCV2-linker-PoIL-2 and r pcDNA3.1/ORF2 were transfected into COS-7 cells by lipofectamine. Indirect immunofluorescent assay (IFA) and porcine IL-2 ELISA assay was used to detect the bioactivities for PCV2 Cap protein and PoIL-2 protein of the expressed recombinant fusion protein (rCap-linker-PoIL-2 protein), respectively. The positive recombinant expression plasmid were extracted and inoculated Balb/c mice and pigs. The immune effects induced by rpcDNA3.1/PCV2-linker-PoIL-2 and rpcDNA3.1/OFR2 was evaluated by ELISA assay for detection PCV2 antibody in Balb/c mice and pigs. The result showed the chimeric gene of PCV2-linker-PoIL-2 and its recombination expression plasmid were successfully constructed. The rCap-linker-PoIL-2 protein was expressed in the cytoplasm of COS-7 cells and displayed the specific immune responses for anti-PCV2 and anti-PoIL-2 antibodies, which indicated the rCap-linker-PoIL-2 protein possessing the duplex bio-activity of Cap protein and PoIL-2 protein. the mice have been immunited by rpcDNA3.1/PCV2-linker-PoIL-2 plasmid and have a clear anti-PCV2 antibodies,after the 3rd immunization,the immunity group antibody is the control group of 3.2 times, and significantly higher than the rpcDNA3.1/OFR2 plasmid control group; rpcDNA3.1/PCV2-linker-PoIL-2 and rpcDNA3.1/ORF2 plasmid can be induced in pigs production of anti-PCV2 immune response, rpcDNA3.1/PCV2-linker - PoIL-2, rpcDNA3.1/ORF2 plasmid immunohistochemistry antibody titers were empty vector control group of 12.46 times and 10.13 times that PoIL-2 in PCV2-linker-PoIL-2 chimeric gene played a very good immune-enhancing effect to rpcDNA3.1/ PCV2-linker-PoIL-2 plasmid immune in mice and pigs.The successful construction of the PCV2-linker-PoIL-2 chimeric gene and its pcDNA3.1 eukaryotic expression plasmid rpcDNA3.1/PCV2-linker-PoIL-2; the recombinant expression plasmid have expressed biological activity fusion protein of Cap-and PoIL-2 in COS-7 cells .The recombinant plasmid can induce mice and pigs have a high anti-PCV2 antibody in mice and pigs and the induced antibody levels were significantly higher than the pcDNA3.1/OFR2 recombinant plasmid.This study for the PCV2 DNA vaccine research laid the foundation.