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| Study on the Construction, Expression and Immune Effect in mice of the Eukaryotic Expression Vector of Porcine Circovirus Type 2 ORF2/Porcine Interleukin-2 Chimeric Gene |
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Abstract Abstract: To exploit the porcine circovirus type 2 (PCV2) DNA vaccine to prevent the PCV2 disease, the recombination chimeric gene of PCV2-linker-PoIL-2 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) method was cloned into pGEM-T Easy vector and subsequently sub-cloned into eukaryotic expression vector pcDNA3.1(+). The positive recombination expression plasmid of pcDNA3.1/PCV2-linker-PoIL-2 (rpcDNA3.1/PCV2-linker-PoIL-2) was selected and transfected into COS-7 cells by lipofectamine. The bioactivities of the expressed recombinant fusion protein (rCap-linker-PoIL-2 protein) in COS-7 cells were tested by the methods of indirect immunofluorescent assay (IFA) for detection PCV2 antigen and porcine IL-2 ELISA assay for detection PoIL-2 protein, respectively. The immune effects in Balb/c mice induced by rpcDNA3.1/PCV2-linker-PoIL-2 and recombinant expression plasmid of pcDNA3.1/OFR2 (rpcDNA3.1/OFR2) were evaluated by ELISA assay for detection PCV2 antibody. The result showed that the chimeric gene of PCV2-linker-PoIL-2 and the rpcDNA3.1/PCV2-linker-PoIL-2 were successfully obtained and constructed, respectively. The rCap-linker-PoIL-2 protein, which was existed in the cytoplasm of COS-7 cells and expressed by rpcDNA3.1/PCV2-linker-PoIL-2, displayed the specific immune response for anti-PCV2 and anti-PoIL-2 antibody by IFA and ELISA assay, respectively, which indicated the rCap-linker-PoIL-2 protein had the duplex bio-activity of Cap protein and PoIL-2 protein. Although the specific antibody response to PCV2 in mice could be induced by being intramuscularly immunized with ether rpcDNA3.1/PCV2-linker-PoIL-2 or the rpcDNA3.1/OFR2, the antibody titer induced by former was much higher than that caused by the latter after the second and third immune. Our study laid the good foundation for further PCV2 DNA vaccine development.
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Received: 04 February 2008
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