Expression Of Porcine Circovirus Type 3 Cap Gene In Insect Baculovirus

Porcine circovirus is a small non-enveloped single-stranded circular DNA virus that that belongs to the genusCircovirus in the family Circoviridae.PCV are divided into four species at present,PCV1,PCV2,PCV3 and PCV4.PCV1 does not cause diseases in pigs,whereas PCV2 causes PCV2-associated diseases for swine,inhibiting the immune system of swine and causing severe secondary infection with other pathogen.It's a globally important pathogen threating to swine health.PCV3 is a newly identified virus isolated from swines that have chronic reproductive problems and clinical signs consistent with porcine dermatitis and nephropathy syndrome.A new circovirus was identified in Hunan province,China,recently.The viral genome shows the highest genomic identity to mink circovirus(66.9%),designated as PCV4,tentatively.The PCV3 genome comprises 2000 nucleotides(nt),virions have an icosahedral structure,non-enveloped,the diameter is 17-20 nm in size,containing three major,inversely arranged open reading frames(ORFs).The ORF1 gene encodes a replicase protein(Rep)that comprises 297 amino acids(aa);The ORF2 gene encodes nucleocapsid protein(Cap),contains 214 aa,is the structural protein of PCV3.While ORF3 gene encodes a protein consisting of 231 aa and its function is not clear.As the only structural protein of PCV3,Cap protein is the main antigen that induction of a specific immune response.The available PCV2 vaccine does not provide effectively protection for PCV3 infection because PCV3 Cap has identity of 30%to the PCV2 Cap,and both Caps don't have common liner epitope.So far,no PCV3 commercial vaccine has been marketed.So,the research and development of PCV3 vaccine is urgent.At present,PCV3 isolation and culture technology is still immature.If the PCV3 subunit vaccine can be prepared based on the Cap protein that produced by high-quality and high-yield,it will provide a powerful support for the prevention and control of PCV3-related diseases.For this reason,this study expressed the PCV3 Cap use the baculovirus expression system,laying the foundation for the development of PCV3 subunit vaccines.1.Construction of PCV3 Cap protein insect baculovirus expression systemFirstly,the PCV3 cap gene was optimized and GP67 singal sequence and KOZAK sequence were added to its 5'-terminus to reach the highest possible level of expression,a 6 ×His-tag was fused to the 3'-terminal end of the Cap to aid protein purification.The optimized ORF2 gene was subcloned into baculovirus transfer vector pFastBacl exploiting Bac-to-Bac baculovirus expression system.The recombinant plasmid pFastBacl-cap was transformed into E.coli DH10Bac competent cells which contain the baculovirus shutter vector(bacmid)and helper vector.The colonies of E.coli DH10Bac containing recombinant bacmid(Bacmid-cap)were collected by blue white selection.Recombinant Bacmid was transfected into sf9 insect cells using LipoInsect transfection reagent to obtain recombinant baculovirus.Expression of the ORF2 gene of PCV3 was confirmed by indirect immunofluorescent assay(IFA)and Western-blotting.The recombinant baculovirus was amplified into a high titer baculovirus,and the virulence value of the recombinant baculovirus was determined to be 108 pfu/ml by plaque test.The optimal multiplicity of infection for Cap protein expression was determined to be 5 and optimal time point is 72 hour after infection.2.Purification of PCV3 Cap protein and preparation of PCV3-LPThen sf9 cells were infected with baculovirus to obtain cap protein.The collected Cap protein was purified by the method of affinity chromatography,size-exclusion chromatography,and sucrose density gradient centrifugation.Finally,exploring the in vitro assembly environment of the cap protein in term of determine the optimal conditions for the cap protein to self-assemble into virus-like particles in vitro.A large number of irregular protein aggregations were observed under the electron microscope,and only a small part could form a VLP-like structure with a diameter of about 20 nm,but the shape was not very regular,and there was no typical icosahedral structure,indicating that the PCV3 Cap protein can assembled VLP,but the optimal conditions for PCV3-LP need to be further explored.3.Construction of PCV3 infectious clonePCV3 isolation and culture technology is immature,the construction of infectious clones provides new ideas for this problem.The infectious cloning vector of PCV3 was transformed into PK15 cells for serial passage after verification by sequencing,and fluorescence was verified by IFA.The results found that after the first generation of transfection,green fluorescence can be detected in PK15 cells transfected with pSK-PCV3 recombinant plasmid and PCV3 circular genome,and the fluorescence is mainly distributed in the PK15 cell cytoplasm,and no green fluorescence is seen in the nucleus.When the continued passage to the 10th generation,the green fluorescence disappeared and the PCV3 virus was not successfully rescued.At the same time,we also constructed an infectious clone of PCV2.After continuous passage,IFA successfully detected fluorescence,indicating that the infectious clone of PCV2 was successfully constructed and PCV2 virus were rescued successfulBased on the characteristics of the PCV3 Cap protein,this study used the Bac-to-Bac baculovirus expression system to express PCV3 Cap protein in insect cells,in order to provide a reference for for the development of a safe and effective PCV3 genetically engineered subunit vaccine.