Construction Of Recombinant Pseudorabies Virus(PRV) Expressing E2 Gene Of Classical Swine Fever Virus(CSFV) And Analysis Of Its Partial Biological Characteristics

Classical swine fever?CSF?is a serious Infectious disease which is harmful to pig industry and must be reported to World Organization for Animal Health?OIE?.There have been many studies on the new swine fever vaccines.Envelope glycoprotein E2 could mediate viral infection and induce the body to produce neutralizing antibodies,as the main structural protein of classical swine fever virus?CSFV?.Therefore,E2 protein of CSFV is an important protective antigen.Besides,E2 protein is a major target protein for the development of innovative live vector vaccines and subunit vaccines.It is shown that E2gene inactivated subunit vaccines using baculovirus expression vector system,E2 gene activated subunit vaccines using adenovirus live vector and pseudorabies virus live vector demonstrated good immune effects in many studies.Pseudorabies?PR or Aujeszky's Disease,AD?caused by pseudorabies virus?PRV?,is an acute infectious disease,which causes high mortality in piglets,reproductive disorders in sows,neural and respiratory symptoms in growing-finishing pig,reduction of performance in boar.The genome of PRV is a linear double-stranded DNA,which is about 145kb,encoding 70 to 100 viral proteins.The genome,being an excellent live virus vector,contains a large number of regions unrelated with virus proliferation,which is available for the insertion of foreign genes.The PRV recombinant live vaccine has good immune effect on the infection of PRV which has similar genetic relationship with the vector.With green fluorescent labeled porcine pseudorabies virus rPRV-BE with TK,gI,gE,US9 and partial gN gene deletion developed by our lab as the parent strain,based on pMD-US6-EGFP-US2,the left homologous arm+gI gene was replaced and CSFV E2 gene with CMV promoter was inserted to construct the transfer plasmid pMD-US6-US7-E2-US2 successfully in the research.Then the PK15 cells were co-transfected with rPRV-BE,the parent strain,and pMD-US6-US7-E2-US2,the transfer plasmid,by lipofection.After multiple rounds of plaque screening and purification,the recombinant pseudorabies virus rPRV-CSFV PE2SC of main immunogenic protein gene named E2 of CSFV?C Strain?expression was obtained.PCR,gene sequencing and indirect immunofluorescence assay?IFA?proved that the construction was correct and E2 protein could be expressed successfully.At the same time,the genetic stability and proliferation characteristics of recombinant virus in PK15 cells were studied.The results showed that the recombinant fragments containing E2 gene of CSFV were 3469bp in size,corresponding to the size of primary recombinant virus,could be amplified by PCR in the 5th,10th,15th and 20th generations of recombinant viruses.The fluorescence of E2 protein was found in the pores of the cells infected by the 20th generation recombinant virus,which indicated that the recombinant virus could be passed on in a stable manner after 20 generations of continuous transmission in vitro.The results of one-step growth curve showed that,compared with wild virus PRV?SX strain?and parent virus rPRV-BE,the recombinant virus r PRV-CSFV PE2SC proliferated faster,reached the peak of virus titer earlier,and the peak virus titer was lower than wild virus.The highest virus titer was 10^6.7TCID₅₀/mL,10^8.0TCID₅₀/mL and 10^7.0TCID₅₀/mL respectively.The results showed that the recombinant virus had good genetic stability and replication ability in vitro.Safety tests were performed on healthy mice,rabbits and piglets,mice and rabbits were vaccinated with dose of 10^4.0,10^5.0,10^6.0,10^7.0 TCID₅₀ into four groups,piglets were vaccinated with dose of 10^6.0,10^7.0 TCID₅₀ into two groups,while the control groups were vaccinated with the same dose of saline solution.During the 14-day observation period after vaccination,no adverse reactions were observed,which indicates that the recombinant virus rPRV-CSFV PE2SC had Low virulence and good safety.The experimental results laid the foundation for the further evaluation of its immune effect and the development of recombinant vaccine with deletion of PRV gene.