Preparation Of Proliferation-defective PCV2 Based On Trans-complementary Cell Line

Since the outbreak of PCVAD caused by PCV2 infection in various parts of China in 2002,it has become one of the most important diseases endangering the pig industry in China.Infection with PCV2 will lead to immunosuppression in pigs,which is prone to secondary infection with other pathogens,resulting in greater economic losses.Vaccine immunization is the main means to prevent PCV2-related diseases.At present,clinically applicated PCV2 vaccine is mainly divided into two categories,inactivated whole virus vaccine and genetic engineered subunit vaccine.Although clinically safe,they can not achieve ideal immune protection.Therefore,many researchers at home and abroad have carried out research on new PCV2 vaccines,including attenuated vaccine,DNA vaccine and so on.This study utilized genetic engineering technology to prepare proliferation-defective PCV2 virus,which lays a foundation for the development of a new type of PCV2 genetic engineering vaccine in the future.1 Construction of p IRES2-CAPopt expression vectorAccording to the codon preference of domestic pigs,the ORF sequence of CAP gene was recoded and artificially synthesized,and cloned into p UC vector by introducing BglⅡsite upstream and Not I site downstream.PUC-CAPopt plasmid and p IRES2-EGFP plasmid were digested by Bgl II and NotⅠto obtain small fragments of CAP and large fragments of p IRES2 vector,respectively,which were ligated with T4DNA ligase and transformed to obtain p IRES2-CAPopt expression vector.The p IRES2-CAPopt was identified by the double digestion,and the result showed that the CAP gene was about 700bp and the PIRES2 vector was about 4000bp,the fragment size was the same as the expected,which means that the expression vector was constructed successfully.2 Construction of CAP trans-complementary transgenic PK15 cell line（CTC-PK15）The endotoxin-free p IRES2-CAPopt plasmid was transiently transfected into PK-15 cells in 96-well plate,and the expression function of CAP in the vector was verified by indirect immunofluorescence assay（IFA）.PK-15 cells in 24-well plate were cultured with different concentrations of G418 for 10-14 days to determine the optimum screening concentration of G418 for PK-15 cells.PIRES2-CAPopt was transfected into PK-15 cells in 6-well plate,cultured with media containing 700ug/ml G418 until no cell death.Monoclonal cells were selected by infinite dilution method and verified by IFA.The results showed that the fluorescence rate of p IRES2-CAPop transient transfected cells reached 10%,indicating that the recoded CAP gene inserted into p IRES2-CAPopt vector was normally expressed in PK-15 cells.After stable transfection of p IRES2-CAPopt into PK-15 cells,CTC-PK15 was achieved via G418 pressure screening and monoclonal screening method.3 Construction of proliferation-defective PCV2 infectious clone p PCV2-M₃Genomic DNA of PCV2 virus was amplified and cloned into p Omni plasmid by PCR-mediated recombination to achieve PCV2 infectious clone p Omni-PCV2.The138,139,140 amino acid codons of CAP gene in p Omni-PCV2 vector were mutated into3 stop codons by site-directed DNA mutagenesis technique to construct the mutant infectious clone p PCV2-M3.The results of PCR identification of transformed clones showed that the PCV2 infectious clone p Omni-PCV2 vector was constructed successfully.The results of p PCV2-M₃infectious clone sequencing showed that three consecutive amino acid codons in CAP gene were successfully mutated into three stop codons TAA,TAG and TGA.4 Rescue of PCV2-M₃replication defect virusCTC-PK15 cells were transfected with infectious clone p PCV2-M₃respectively,then blindly passed for 12 passages under G418 maintaining concentration.PCR amplification showed that virus DNA increased passage by passage,which proved the success rescue of proliferation-defective virus PCV2-M₃.The DNA of PCV2 virus with known titer(TCID₅₀=10⁵)was 10-fold gradiently diluted to 10^-6,and each dilution DNA was used as template for PCR amplification.The virus titer of 12th blind-passage of PCV2-M₃virus was estimated by PCR amplification of its 10-fold gradiently diluted DNA and compared with that of PCV2.The results showed that the maximum dilution of the positive amplification of PCV2 viral DNA was 10^-5(corresponding to known titer of 10⁵TCID₅₀),and the maximum dilution of the positive amplification of PCV2-M₃viral DNA was 10^-4,because the dilution of PCR templates and the dilution of firus in IFA both were 10-fold gradient dilution,so it can be inferred that the TCID₅₀of PCV2-M₃is about 10⁴.5 Verification of proliferation defects of proliferation-defective PCV2-M₃Rescued PCV2-M₃virus was used to infect PK-15 cells and CTC-PK15 cells respectively and blindly passed for three passages.The virus DNA was extracted from the lysate of the two kinds of cells.The proliferation defect of PCV2-M₃virus in normal PK-15 cells was verified by specific PCR.The results showed that the lysate of P1-3 of CTC-PK15 cells infected were positively amplified,but only the lysate of P1 of PK-15cells infected was positively amplified and no amplified bands were found in PCR with P2 and P3 PK-15 cell lysates.It’s indicated that PCV2-M₃virus is infectious to normal PK-15 cells but can not produce offspring virus to infect next generation of PK-15 cells.CTC-PK15 cells were infected with PCV2-M₃virus and passed for 30 passages and the virus was harvested every 10 passages.After infecting PK-15 cells with the harvested virus,the reverse mutation of PCV2-M₃virus was verified by IFA.The results showed that the IFA of PK-15 cells infected the P30 virus was still negative,demostrating that no reverse mutation took place in 30 passages.It is indicated that the possibility of reverse mutation of the three stop codons of PCV2-M₃virus designed and constructed in this study is very low and it is a candidate vaccine with excellent safty.