Construction And Immunogenicity Of TK~-/gE~-/gG~- G-recombinant Pseudorabies Virus Expressing Porcine Circovirus Type 2 Capsid Protein

Porcine circovirus type 2?PCV2?is associated with several porcine disease syndromes,known as porcine circovirus associated diseases?PCVAD?while postweaning multisystem wasting syndrome?PMWS?has the most serious clinical manifestation.In China,PCV2 is prevalent in lots of swine herds,which seriously endanger the health condition of swine herds and cause tremendous economic losses to the swine industry.Porcine pseudorabies?PR?is an acute infectious disease caused by pseudorabies virus?PRV?.Newborn piglets infected with PRV could lead to high mortality.Sows infected with PRV can cause reproductive disorders.It is difficult to eradicate PRV from a swine farm once infected with it,which will seriously affect the economic benefits of swine farms.PCV2 Capsid protein is the major protective antigen which can stimulate the body's immune syetem to produce specific neutralizing antibodies against PCV2.Therefore,PCV2-Cap protein is an ideal target antigen for the development of the genetic engineering vaccine.Nowadays,PRV as an expression vector to express exogenous protective antigen has been a hot topic in the field of genetic engineering live vector vaccine.In order to eludicate the genetic background of the PRV-JF strain,the U_L44?gC?gene was sequenced and analyzed to illustrate the genetic envolution relationship among PRV-JF,PRV-SC and PRV epidemic variants in recent years.The gC amino acid sequences of the six PRV isolates and other thirty sequences of PRV strains available in GenBank were used for analysis and comparison.The results suggested that the six isolates and PRV variants isolated in China since 2011 belonged to the same branch,shared 98.7%to 100%identity with PRV variants,while only 93.2%to 94.0%identity with the classic strain PRV-SC.A hypervariable region located in the 52^th to70^th amino acid was found on the gC protein for the six PRV isolates,especially the insert sequences of seven amino acids(⁶⁴AAASTPA⁷⁰).Although the PRV-JF strain isolated in 2008,it was in the same branch with the PRV variants isolated after 2011,not belonged to the PRV classic strains.The PRV-JF strain as the parental vector to construct recombinant virus could contribute to control the PRV variants.For improving the PCV2-Cap expression level of the recombinant virus r PRV-JF-TK^-/gE^-/2Cap⁺,the non-essential gene U_S4 was selected as a new insertion site to insert another PCV2-Cap expression cassette.In this study,a new recombinant virus was constructed by traditional homologous recombination.Co-transfect the genome of rPRV-JF-TK^-/gE^-/2Cap⁺with pMD-EGFP-LR?gG?into PK-15 cells with calcium phosphate transfection kit.The U_S4?gG?gene could be replaced by EGFP expression cassette based on the intracellular homologous recombination.With twenty circles of plaque purification,the recombinant virus was identified by PCR.Co-transfect the genome of rPRV-JF-TK^-/gE^-/2Cap⁺/g G^-/EGFP⁺with the transfer plasmid pMD-Cap-LR?gG?into PK-15 cells with calcium phosphate transfection kit.The EGFP expression cassette was replaced by PCV2-Cap and then purify the recombinant vius with six rounds of reversed screening without EGFP.In order to distinguish the new constructed virus from rPRV-JF-TK^-/gE^-/2Cap⁺,the C terminal of PCV2-Cap protein was inserted with the flag tag.The expression of new PCV2-Cap-flag expression cassette was identified by IPMA and IFA with the anti-PCV2 mAb and anti-flag mAb.In order to verify the immunogenicity of r PRV-JF-TK^-/gE^-/g G^-/3Cap⁺in vivo,six to seven weeks old healthy piglets were used to carry out the vaccination and challenge test.Based on the observation of clinical symptoms and the detection of anti-PRV or PCV2 antibodies level,the immunogenicity of the recombinant virus expressing PCV2-Cap in pigs could be identified.Anti PRV-gB antibody could be detected by IDEXX Co.PRV gB kit,and the antibody levels gradually increased.However,the antibody to PCV2 couldn't be detected by IPMA method post-vaccintion 28 days.The recombinant virus expressing PCV2-Cap protein did not stimulate the immune response in piglets.The PRV JF strain as a vector to express heterologous protein for the development of genetic engineering live vector vaccine needs further exploration.A recombinant virus rPRV-JF-TK^-/gE^-/gG^-/3Cap⁺with three genes deletion to express PCV2-Cap protein was successfully constructed in this study.The PCV2-Cap-flag expression cassette could be expressed in vitro experiment;however,the immunized piglets with recombinant virus couldn't induce the humoral immune response to PCV2-Cap.The reasons need to further study the immunogenicity influence factors for the recombinant PRV as expression vector to develop genetic engineering live vector vaccine.