Construction And Immune Efficacy Of Recombinant PRV Strainsof CS Epitope Region Of PEDV S1 Gene And Porcine IL-6

Porcine epidemic diarrhea(PED)was an acute,highly contagious and severe intestinal disease caused by the porcine epidemic diarrhea virus(PEDV).Before 2010,PED was sporadic in all parts of my country,and the incidence and mortality of pigs were very low.At the end of 2010,a new variant strain of PEDV appeared,and many pig farms in China had a severe outbreak of PED,which has a high morbidity and high mortality rate for pigs,especially the fatality rate of infected piglets was as high as 100%,causing huge economic losses.At the end of 2011,the emergence of variant strains of pseudorabies virus(PRV)also brought new challenges to the purification of pseudorabies(PR).It has been reported that the use of PRV as a live vector to express the epitope regions of other viruses can achieve the effect of bivalent vaccines.The classic vaccines of PEDV and PRV cannot provide complete protection to the current circulating strains.Therefore,new vaccines for the current epidemic variants of PEDV and PRV are in urgent need of development.The neutralizing epitope of PEDV is mainly located in S1,which has been confinned that there are 4 main neutralizing epitopes(aa 499-638,748-755,764771 and 1 368-1 374)on the surface of PEDV S protein.The CS region(aa 499789)contains the first three main neutralizing epitopes of the S protein.In order to obtain the recombinant PRV virus expressing the PEDV neutralizing epitope region CS,this experiment cloned the PEDV CS epitope region gene and inserted it into the PRV transfer plasmid pEG through the BamH I restriction site to obtain the recombinant transfer plasmid pEG-CS.Then it was transfected with rPRV NY-gE/gI-/TK-strain into porcine testis(ST)cells to undergo homologous recombination.After plaque purification,the recombinant virus rPRV-CS-EGFP+ with green fluorescent tag was obtained.Then the CRISPR/Cas9 knockout system(PX459gRNA1-EZ-gRNA3-EGFP)was used to remove the EGFP fluorescent label,and the recombinant virus rPRV-CS was successfully obtained.The PCR test showed that the CS gene was successfully inserted into the PRV genome.RT-PCR and Western-blot and indirect immunofluorescence tests showed that the neutralizing epitope region of CS can be transcribed and expressed.Interleukin 6(IL6)is a multifunctional cytokine that can stimulate the proliferation and activation of T cells and the differentiation of B cells to participate in adaptive immunity.IL6 was widely used in vaccine adjuvants,tumor adjuvant therapy,disease diagnosis and other fields,and has good application prospects.In order to obtain a recombinant PRV virus simultaneous expression PEDV neutralizing epitope region CS and porcine IL6,and to explore the effects of IL6 as an immune adjuvant,the pig IL6 gene was amplified from pig blood through extracts total RNA in this experiment,and then BamH I and Hind III restriction sites were added to both end of the primers,and it was inserted into the mammalian eukaryotic expression plasmid pBApo-EF1α_Pur_DNA.At the same time,the IL6 sequence was inserted into the mammalian eukaryotic expression plasmid pBApoEF1α_Pur_DNA.The KozaK sequence(GCCACC)was previously introduced to enhance the expression of IL6 protein.Then use high-fidelity enzyme to amplify the IL6 expression cassette from the vector,and insert into the recombinant transfer plasmid pEG-CS through the Bstz17I restriction site to obtain the recombinant transfer plasmid pEG-CS-IL6.Which has homologous recombination with rPRV NY-gE-/gI-/TK-strain,the recombinant virus rPRV-CS-IL6-EGFP+was purified by plaque.Subsequently,the recombinant virus rPRV-CS-IL6 was successfully obtained by removing the EGFP fluorescent label.The PCR test showed that the CS gene and IL6 expression cassette were successfully inserted into the PRV genome.RT-PCR and Western-blot and indirect immunofluorescence tests showed that the neutralizing epitope region of CS and IL6 can be transcribed and expressed.In this study,the growth characteristics,TCID50,genetic stability of recombinant viruses rPRV-CS and rPRV-CS-IL6 on ST cells,PK-15 cells,Vero cells,and IPEC cells,and the safety and immune efficacy in mice were tested.The results showed that the TCID50 of the recombinant viruses rPRV-CS and rPRV-CS-IL6 measured on ST cells were 106.5/0.1mL and 106-875/0.1mL,respectively.The results showed that ST cells were more suitable for the propagation and culture of recombinant viruses rPRV-CS and rPRV-CS-IL6.The growth characteristics of the recombinant viruses rPRV-CS and rPRV-CS-IL6 were similar to those of the parent strain rPRV NY-gE-/gI-/TK-virus,with good genetic stability,safe for mice.They can induce mice to produce specific immune responses against PRV and PEDV,protect mice from the virulent and lethal PRV attack.In addition,the level of antibodies produced by the recombinant virus rPRV-CS-IL6 group was slightly higher than that of the recombinant virus rPRV-CS group,indicated that IL6 cytokines have a certain immune-enhancing effect and can be used as a vaccine adjuvant.In summary,this study successfully constructed two recombinant viruses rPRV-CS and rPRV-CS-IL6 by using the rPRV NY-gE-/gI-/TK-strain as the parent strain,and explored their biological characteristics and immune efficacy in mice.This laid a certain foundation for the next step of related immune research on pigs.The recombinant viruses rPRV-CS and rPRV-CS-IL6 were expected to be candidate strains for dual attenuated vaccines against PEDV and PRV,providing a powerful tool for the effective prevention and control PED V and PRV,but also laid the theory of genetic engineering live vector vaccine development and technical support.