Baculovirus Expression And Antigenicity Of Multiple Epitopes Of Classical Swine Fever Virus Glycoprotein E2

Classical swine fever(CSF)is a highly contagious viral disease of domestic pig and wild boar caused by C lassical swine fever virus(CSFV),and causes large economic losses for pig industry worldwide.Currently,vaccination is still the main method of prevention and control for classical swine fever.The most effective CSF vaccine is the attenuated vaccine which obtained by artificial domestication of CSFV virulent strains,and has been used in many countries and regions to achieve the purification and eradication of CSF.However,there are still some risks in the use of attenuated vaccines,such as the risk of transforming attenuated strains to virulent strains and the immune tolerance of piglets caused by vertical infection.Therefore,some countries and regions have banned the use of attenuated vaccines in production.The CSF attenuated vaccine lacks immunological markers and cannot distinguish between infected and immunized animals.It makes the purification and eradication of CSF difficult in our country.Glycoprotein E2 is the main antigen of CSFV,and is an ideal candidate protein for the development of the CSF subunit vaccine.In this rescerch,we added same linear epitope into the glycoprotein E2 of CSFV C strain to design a recombinant protein r E2,in order to improve the immunogenicity of glycoprotein E2.Artificially synthesize the nucleotide sequence of r E2.The baculovirus gp67 signal peptide and the r E2 gene were cloned into the transfer vector p Fast Bac Dual to obtain the transfer vector p FBD-gp67sg-r E2.Similarly,cloning the baculovirus gp67 signal peptide and the E2 gene into transfer vector p Fast Bac Dual and obtain the transfer vector p FBD-gp67sg-E2.The recombinant vector p FBD-gp67sg-r E2 and p FBD-gp67sg-E2 were confirmed by PCR and digestion.The recombinant vector p FBD-gp67sg-r E2 and p FBD-gp67sg-E2 were transformed into DH10Bac to obtain the recombinant Bacmid r Bac-gp67sg-r E2 and r Bac-gp67sg-E2.The r Bac-gp67sg-r E2 and r Bac-gp67sg-E2 were confirmed by PCR.Then Sf9 cells were transfected with r Bac-gp67sg-r E2 and r Bac-gp67sg-E2 to obtain the recombinant virus Ac-gp67sg-r E2 and Ac-gp67sg-E2respectively.Western blot and indirect immunofluorescence assays were used to detect the expression of r E2 and E2 by Sf9 cells infected with Ac-gp67sg-r E2 and Ac-gp67sg-E2.The results showed that the recombinant virus Ac-gp67sg-r E2 and Ac-gp67sg-E2 were successfully constructed.The recombinant virus Ac-gp67sg-rE2 infected High Five cells as a n MOI of 0.1,0.5,1,2,respectively.The concentration of r E2 protein in cultures at 48 h,72 h,96 h and 120 h post infection was detected by Western blot semi-quantitative method.The results showed that the expression of r E2 protein could be detected in the cell cultures of different infectious doses at 48 h post infection.The concentration of r E2 protein in cultures reached to the highest at 96?120 h post infection.When MOI=2,the r E2 concentration in culture was reached to the highest,350?g/m L;at 96?120 h after infection,the cells began to lyse,and the recombinant protein in cell began to degrade.The results indicated that the addition of hydrophilic linear epitope and gp67 signal peptide could greatly increase the expression yield of r E2.The rE2 and E2 with oil adjuvant were vaccinated 2 months old piglets,respectively.The immune group was as follows:Group 1 was vaccinated with r E2,Group 2 was vaccinated with E2,and Group 3 was injected with PBS as blank control.Each group was given twice vaccination,each vaccination interval of 3 weeks.Detection of serum CSFV E2 antibody of each piglet in 7 d,14 d,21 d,28 d,35 d post vaccination with ELISA.Two weeks after the second vaccination,piglets were challenged with 5×10⁵ TCID₅₀ CSFV shimen strain.The results showed that both r E2 and E2 could induce high levels of CSFV E2 antibody after the second vaccination;piglets in Groups 1 and 2 had a survival rate of100%after challenge.The results indicated that the r E2 protein we designed and expressed in this study has good immunogenicity.