Researches On Packaging-defective PCV2 As DNA Vaccine Vector Expressing GP5 Of PRRSV

DNA vaccines have been a focus of researches on genetically engineered vaccines around the world since 1990s,however,compared to traditional vaccines,the efficacy of DNA vaccines remained poor because the genes of target antigen can’t effectively enter host cells and can’t replicate,and thus the target gene is expressed at very low level.To address this matter,this study proposes a technical solution for a self-replicating DNA vaccine based on packaging-defective virus.PCV2 virus was selected as the vector for construction of the recombinant virus,the Cap gene that is essential for packaging of PCV2 virus but is not needed for genome replication was selected to be replaced by the target gene,and a transgenic cell line that expresses the Cap protein was used for preparation of the packaging-deficient recombinant virus.The target gene can effectively enter host cells through virus infection and replicate along with the virus genome,and is thus expected to be expressed at high level in host cells.Meanwhile,the recombinant virus would not be able to produce progeny virus particles due to lack of the Cap protein,so it would be safe despite active DNA replication and protein expression.The study included the following aspects:1.Construction of the infectious clone of packaging-defective PCV2-GP5Genome DNA of the PCV2 virus was amplified by PCR and cloned into the p Omni plasmid by PCR-mediated recombination to achieve PCV2 infectious clone p Omni-PCV2.The GP5 gene fragment was obtained from PRRSV live vaccine with reverse transcription and amplified by PCR,then cloned into p Omni-PCV2 plasmid by means of PCR-mediated recombination,resulting in infectious clone p Omni-PCV2^△CAP-GP5plasmid.Sequencing results showed that the 561bp GP5 gene fragment was 100%identical to what’s recorded in Gen Bank（accession number:AEI72610.1）,and perfectly replaced part of the Cap gene,forming a in-frame open reading frame（ORF）.2.Rescue of the PCV2-GP5 packaging-defective virusEndotoxin-free p Omni-PCV2^△CAP-GP5plasmid was transfected into transgenic CTC-PK-15 cells that express the Cap gene of PCV2 to allow packaging of the defective virus.The transfected cells were cultured under selection pressure from G418at maintenance concentration for 3 blind passages,and medium containing virus particles were harvested from each passage.PCV2-GP5 harvested from the third passage was tested by PCR for presence of the GP5 gene fragment,using untransfected CTC-PK-15 cell as negative control and p Omni-PCV2^△CAP-GP5vector as positive control.The expected 561bp band was observed in the test group and positive control,but not in the negative control group,suggesting successful rescue of the recombinant PCV2-GP5virus.3.Verification of the proliferation-deficiency of the PCV2-GP5 virusPCV2-GP5 virus DNA was extracted from passages in CTC-PK-15 cells and specific PCR was used to evaluated the proliferation of the virus in CTC-PK-15 cells.CTC-PK-15 and PK-15 cells were infected with PCV2-GP5 virus respectively,then cultured for 3 passages.The results show that the recombinant virus PCV2-GP5 can continually proliferate in CTC-PK-15 cells.The maximum dilution of the positive amplification of PCV2-GP5 viral DNA was 10^-4with the template infinite dilution method,while the highest dilution of the positive amplification of PCV2 viral DNA(TCID₅₀=10⁵)was 10^-5,from which it can be inferred that the TCID₅₀of PCV2-GP5 is about 10⁴.The GP5 gene could be detected by means of PCR in P2 and P3 of CTC-PK-15 cells but not in P2 or P3 of PK-15 cells,indicating that the PCV2-GP5virus can proliferate in CTC-PK-15 cells but not in PK-15 cells.The GP5 gene could be detected in infected PK-15 cells（P1）means that PCV2-GP5 virus can infect PK-15cells.4.Verification of GP5 expression in PK-15 via PCV2-GP5 packaging-defective virusPK-15 cells were infected with PCV2-GP5 packaging-defective virus,then total RNA of the infected cells was isolated for detection of GP5 m RNA expression,using uninfected PK-15 cells as negative control.Both RNA samples had D₂₆₀/D₂₈₀values>1.8,indicating that the samples were pure and free of protein contamination;RNA electrophoresis of the test sample also showed 3 distinct r RNA bands（28s r RNA,18s r RNA,and 5s r RNA）suggesting good RNA quality.RT-PCR showed a band at the expected size of GP5 fragment in the test group but not in the negative control group,indicating successful m RNA expression of GP5 in PK-15 cells through PCV2-GP5packaging-defective virus,which makes it possible to proceed immunization trial in animal in future researches.