Preparation Of Replication-deficient PCV2 Virus Expressing Chimeric Epitope Of PRRSV GP3-5

Porcine circovirus-related disease(PCVAD)is a variety of pig diseases caused by porcine circovirus type 2(PCV2),including: weaned piglet multiple system failure syndrome(PMWS),porcine dermatitis nephritis syndrome(PDNS),porcine respiratory tract Disease Syndrome(PRDS),etc.Porcine Reproductive and Respiratory Syndrome(PRRS)is caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV).A large number of studies have confirmed that both PCV2 and PRRSV are important pathogens that cause immunosuppression in pigs.PCV2 and PRRSV infection often lead to immune failure and increased incidence of other infectious diseases in pigs.Therefore,PCV2 and PRRSV are considered two viruses causing the greatest economic loss in pig industry.Immunological control(ie vaccination)is the most effective way to control PCVAD and PRRS.There are currently only two kinds of commercial PCV2 vaccines: inactivated vaccines and subunit vaccines based on Cap protein.There is no attenuated live PCV2 vaccines commercially available.The former two vaccines have poor effect to induce cellular immunity,as a result,immune adjuvant and multiple immunizations are necessary.Inactivated vaccines and live attenuated vaccines against PRRSV are both available but their clinical effects are not perfect.Therefore,reverse genetic manipulation and genetic engineering techniques are used to prepare a replication-defective PCV2 virus expressing the chimeric epitope of PRRSV GP3-5 in this study.The prepared replication-defective live PCV2 virus will have the advantage of higher safety than attenuated live vaccine.Moreover,it can express chimeric epitope of PRRSV GP3-5 after intering host sells,and as a result,has potential to be used as subunit vaccine against PRRSV.The main research contents and results are as follows:1.Construction of Cap trans-complementation transgenic PK15 cell line:p CMV-CAP-opt was extracted with an endotoxin-free kit.The result of Transient transaction of PK15 cell with p CMV-CAP-opt and RT-PCR verification showed that Cap gene in the plasmid can be expressed in the dell.After digestion with endonuclease Afl II and gel-purification,p CMV-CAP-opt linear fragment was transfected into PK15 cell.Transgenic PK15 cells were obtained by repeated pressure screening with G418.Cap trans-complementation transgenic PK15 cell line was obtained by monoclonal culture and screening.IFA verification showed that the Cap gene was expressed in the transgenic PK15 cells,which measns that CTC-PK15 cell line was successfully constructed.2.PCV2-GP3-5 infectious clone construction: The artificially synthesized GP3I-GP5I-GP5 IV chimeric epitope gene was inserted into the p Omni-PCV2 plasmid using PCR recombination cloning technology.The 180 bp GP3-5 chimeric epitope sequence was inserted into the Cap gene of PCV2,replacing the 122-183 AA of Cap gene.174 bp sequence in Cap is replaced,so the genome of themutant virus is enlarged by 6bp.The specific PCR amplification of the GP3-5 chimeric epitope gene confirmed that the p Omni-PCV2-GP3-5infectious clone was successfully constructed,and the sequencing results also showed that the p Omni-PCV2-GP3-5 infectious clone was successfully constructed without base mutations.3.PCV2-GP3-5 virus rescue: the p Omni-PCV2-GP3-5 was digested with endonuclease Sal I to recover the PCV2-GP3-5 genome fragment.T4 DNA ligase was used to circularize the genome DNA before transfected into CTC-PK15 cells.After the cells were blinded for 3generations,the third generation virus was collected to infect CTC-PK15 cells and PK15 cells respectively.The cells were blindly passed for 3 generations and then the sixth generation viral DNA was extracted.PCV2 gene and GP3-5 gene specific PCR detection and rescue mutant virus.The result of specific PCR of PCV2 genome and GP3-5 chimeric epitope gene showed that the mutant virus PCV2-GP3-5 was succefully rescued.4.Verification of the replication defect of PCV2-GP3-5 virus: Successfully rescued PCV2-GP3-5 virus in CTC-PK15 was transferred into T75 cell vial and continuously propagated.The TCID50 of the PCV2-GP3-5 virus was determined by PCR template infinite dilution method as 103.The PCV2-GP3-5 virus was used to infect CTC-PK15 cells and PK-15 cells respectively,the progeny virus was harvested 48 hours after infection to infect new cells for 3 passages respectively.Virus DNA of 3 passages was extracted and verified by specific PCR of PCV2 genome.The results showed that PCV2-GP3-5 virus continuously multiplies in CTC-PK15 cells to produce progeny virus,but no progeny virus of PCV2-GP3-5was detected in PK15 cells.Detection of GP3-5 expression of PCV2-GP3-5 virus in PK-15 cells: Normal PK15 cells was infected with PCV2-GP3-5 virus,and non-infected PK15 cells not infected with any virus were used as a blank control.The total RNA of the cells were extracted 36 hours after infection for RT-PCR amplification.The results showed that the GP3-5 chimeric epitope gene can be expressed in normal PK15 cells infected with PCV2-GP3-5 virus,indicating that the PCV2-GP3-5 virus can be used as a PRRSV subunit vaccine carrier.To sum up,The replication-deficient PCV2-GP3-5 live virus prepared in this study is safer than traditional attenuated live virus vaccine because it can not replicate and pass on in PK15 cell.Moreover,it has the potential as bivalent vaccine against PCV2 and PRRSV.