Soluble And Highly Expressed Baculovirus System PCV2 Cap Protein And PCV3 Cap Protein And Immunogenicity Research

Porcine circovirus can infect weaned piglets and fattening pigs,and can cause a variety of diseases related to porcine circovirus.It is a huge threat to the pig industry in the world today,which seriously affects the development and progress of pig industry.According to the differences in the antigenicity,pathogenicity,and genetic homology of the serotypes of the virus to pigs,Porcine Circoviruses are classified intoPorcine Circovirus type 1,Porcine Circovirus type 2,and Porcine Circovirus type 3.PCV1 is known to be non-pathogenic,and PCV2 is the main cause of porcine circovirus-as sociated diseases(PCVADs).The most serious of these is Postweaning multisystemic wasting syndrome,(PMWS),which is widespread in China and the world,and has become a common and frequently-occurring disease.PCV3 infection can cause dermatitis nephrotic syndrome in pigs,reproductive disorders in sows,and so on.Multi-system inflammatory reactions occur in diseased piglets,leading to organ dysfunction,which seriously affects the development of pig farming worldwide.However,the amino acid homology of the capsid proteins of PCV2 and PCV3 is as low as 30%,and there is almost no cross-immunity protection between the two viruses.Currently,there are no vaccines and drugs that can prevent and control both PCV2 and PCV3.In this study,the baculovirus expression system was used to express the Cap proteins of PCV2 and PCV3,which will not only help the development of PCV2 and PCV3 vaccines,but also provide proteins for the development of diagnostic kits.Using this system to simultaneously express fusion proteins and perform subsequent purification and vaccine development is safer and more effective than traditional methods,and has important significance for the control of virus-related diseases and prevention of PCV infection.This study is divided into the following areas:Analysis of genetic evolution of Porcine circovirus type 2 in Shandong Province from 2013 to 2018: To reveal the genetic evolution of PCV2 in Shandong Province and analyze representative PCV2 dominant strains,we analyzed 40 cases uploaded to Gen Bank in Shandong Province from 2013 to 2018.Complete PCV2 genome sequence was constructed using MEGA V5.0 software.Phylogenetic tree analysis showed that the evolution of PCV2 genotypes in Shandong Province was the coexistence of 3 genotypes(2a,2b,2d),and PCV2 d was the main epidemic strain.Analysis of the amino acid sequences encoded by ORF2 of different genotypes revealed that specific amino acid sites existed in different genotypes,mainly distributed between amino acids 81-160 encoded by ORF2.Expression of PCV2 Cap protein and PCV3 Cap protein in baculovirus expression system: Insect cell-baculovirus expression system is used to express PCV2 Cap and PCV3 Cap simultaneously,and a section of apicin signal peptide sequence is added to the front of the recombination sequence to facilitate its secretion and expression.Double promoters were used to express the recombinant baculovirus,and indirect immunofluorescence results showed that the recombinant baculovirus can specifically bind to mouse anti-PCV2 Cap and mouse anti-PCV3 Cap monoclonal antibodies,respectively.Genomic PCR identification results showed that it had been successfully amplified and corresponding a specific product with the size of the target fragment;further Western-Blot identified a protein band with a molecular weight of about 70 k D;this experiment successfully expressed a protein containing the PCV2 Cap gene and the PCV3 Cap gene using a baculovirus expression system.Efficient expression provides new methods.Efficient expression of recombinant baculovirus: Optimize the expression conditions to promote efficient expression of recombinant proteins.According to the measured results of P4 recombinant baculovirus titers,sf9 cells were infected with MOI = 1,0.5,and 0.1,respectively,and the virus liquid was harvested by setting five time periods of 24 h,48h,72 h,96h,and 120 h.The experimentally optimized fusion protein acquisition conditions were: MOI = 0.1 to inoculate 2.5 × 106 cells/m L of sf9 cells,and the fusion protein was harvested 4 days after inoculation,with an average expression of 0.7 mg/m L and a maximum expression of 0.85 mg/m L.This method can express PCV2 Cap and PCV3 Cap fusion protein simply and efficiently,which is2-8 times the expression amount of other methods at present,which greatly increases the expression efficiency.It is established for the further development and production of PCV2 and PCV3 duplex subunit vaccine a good theoretical foundation.