Construction Of Recombinant Pseudorabies Virus Expressing GP3/GP5/M Genes Of Porcine Reproductive And Respiratory Syndrome Virus(PRRSV) NADC30-like

Porcine reproductive and respiratory syndrome(PRRS),also known as blue ear disease,is an acute contact infectious disease caused by Porcine reproductive and respiratory syndrome virus(PRRSV).The symptoms of PRRSV were reproductive failure in sows and respiratory disease in newborn piglets.At present,PRRSV NADC30-like strain has caused great harm to the pig industry and there was no effective vaccine to prevent and control it.Studies have shown that these GP5,GP3 and M three genes of PRRSV were associated with protective immunity.And interferon-gamma(IFN-γ)and interleukin-18(IL-18)play an important role in cellular immunity and have a promising application as molecular immune adjuvants.Pseudorabies(PR),also called Suid alphaherpesvirus 1 or Aujeszky’s Disease,is caused by pseudorabies virus(PRV).It is characterized by neurological symptoms and death in piglets,respiratory diseases in fattening pigs and reproductive failure in pregnant sows.The PRV Bartha-K61 vaccine has been used in the production for many years,and its clear genetic background ensures its safety as a viral vector.The p Belo BAC11-Bartha-K61 and its rescue virus were successfully constructed in our laboratory.In this study,on the basis of this plamform,Red/ET recombination combined with site-specific recombination was used to replace PRV TK,g G genes,insert PRRSV NADC30-like GP3-GP5-M genes and IL-18-IFN-γ in infections clone p Belo BAC11-Bartha-K61.Recombinant viruses were successfully resuce and their biogical properties were analysis.The results are as follows:1.In this study,transfer vectors were constructed which containing PRV TK or g G homologous arms.GP3-GP5-M gene of PRRSV were inserted to the three promoters downstream(TK ori promoter,CMV promoter,CAGGS promoter),and IL-18-γ were inserted to the PRV g G promoter downstream.The amplified linear fragments were electrically transformed into E.coli GB2005-dir which expressing Rec ET recombinant enzyme,forming target plasmids through linear plus homologous recombination.The correct clones were obtained through resistance selection,PCR identification and sequencing verification.These correct clones were named p BR322-spect-TK-ori-PRRSVNADC30-like-GP3-GP5-M-lox P-genta,p BR322-spect-TK-CMV-PRRSV-NADC30-likeGP3-GP5-M-lox P-genta,p BR322-spect-TK-CAGGS-PRRSV-NADC30-like-GP3-GP5-Mlox P-genta,p BR322-spect-g G-IL-18-IFN-γ-lox P-kan.2.In this study,the TK gene of Bartha-K61 was first replaced,and then the g G gene.Through the preset Xho I and Afl II cleavage sites in the transfer vectors,the linear fragments were obtained by enzyme digestion and then electrically transformed into E.coli GB2005-red to perform Linear-circular homologous recombination.PRRSV-NADC30-like-GP3-GP5-M and IL-18-IFN-γ were respectively substituted TK and g G genes.Next,resistance elimination was achieved by inducing the expression of specific recombinant enzymes Cre.Three recombinant plasmids were successfully constructed and recombinant viruses were rescue.These three recombinant viruses were named r Bartha-K61-△TK/ori-GP3-GP5-M-△g G/IL-18-γ,r Bartha-K61-△TK/CMV-GP3-GP5-M-△g G/IL-18-γ,r Bartha-K61-△TK/CAGGS-GP3-GP5-M-△g G/IL-18-γ.3.In this study,the proliferation characteristics,gene transcription level and genetic stability of the three recombinant viruses on PK-15 cells were analyzed.The results of three viruses growth curve showed that the overall growth curve were consistent with that of the Bartha-K61 and r Bartha-K61,reaching the peak at 36 h with no significant difference(p>0.05).The gene transcription level of three recombinant viruses was high and could express the foreign protein IL-18.The recombinant viruses were passed on PK-15 cells continuously for 15 generations,all of which appeared lesions and the foreign genes could exist stably.It suggested that the three recombinant viruses have great genetic stability and replication ability in vitro.In this study,three recombinant pseudorabies viruses expressing PRRSV NADC30-like GP3-GP5-M genes and IL-18-IFN-γ were successfully constructed,providing ideas for the future development of candidate vaccines against PRV and PRRSV.