Study On The Suitable Sites In Pseudorabies Virus For Insertion Of Porcine Epidemic Diarrhea Virus S Gene

China has a large number of pigs,and the scale and intensive degree of pig breeding is getting higher.The emergence of pseudorabies virus（PRV）variant and porcine epidemic diarrhea virus（PEDV）variant has caused serious losses in China’s pig industry,the inactivated vaccine and attenuated vaccine developed by Bartha K61 strain and CV777strain are difficult to provide strong enough immune efficacy,so it is of great significance to develop the combined vaccine against these two diseases.PRV belongs to herpesvirus,and its gene deletion attenuated live vaccine can prevent porcine pseudorabies.Therefore,based on PRV attenuated strain as the vector and inserting the protective antigen of swine diseases pathogens to construct the live vector vaccine can not only prevent porcine pseudorabies,but also express foreign gene efficiently and stimulate the production of immunity.The S protein encoded by PEDV S gene contains multiple antigen recognition epitope,which can mediate the production of protective antibodies by the host.It is the preferred target protein for the generation of live vector vaccine.Therefore,the construction of PRV recombinant PEDV mutant S protein bivalent live vector vaccine is of great significance for the prevention and control of PRV mutant and PEDV mutant infection alone or mixed infection.Insertion site can play a crucial role for the expression of foreign genes.In PRV genome,some non-coding regions are unnecessary for virus replication,especially the non-coding regions in downstream of the two open reading frames.The insertion of foreign genes in these non-coding sites may have little effect on the original biological characteristics of the virus,and can effectively express foreign genes.Therefore,in this study,bacterial artificial chromosome technology was applied for insertion of the S gene of PEDV mutant into the non-coding regions between UL11 and UL10,UL35 and UL36,UL46 and UL27 of pseudorabies virus mutant with dual deletion of TK&g E genes,and PRV-S^11-10,PRV-S^35-36,PRV-S^46-27 were constructed.The biological characteristics of the three recombinant viruses and their parent strains were determined by checking the effect of the insertion sites of PEDV S gene on the growth characteristics of PRV in vitro,and detecting the expression and transcription level of S protein of the three groups of recombinant viruses,so as to provide a foundation for further construction of PRV live vector multiple vaccines.1 Construction of recombinant pseudorabies virus with insertion of porcine epidemic diarrhea virus S geneUsing BAC technology and Red two-step homologous recombination method,the expression cassette of PEDV S gene was inserted into the non-coding region of BAC^{PRVΔTK/g E/g I} genome between UL11 and UL10,UL35 and UL36,UL46 and UL27respectively.Three groups of recombinant BAC were constructed:BAC^{PRV-S（11-10）},BAC^{PRV-S（35-36）},BAC^{PRV-S（46-27）}.Then the DNA of recombined BACs,DNA fragments PRV g I and partial g E gene were cotransfected into ST cells to rescue the virus through liposome transfection,and the mini-F sequences in the recombined BAC was replaced simultaneously.After purification and identification,the recombinant viruses were obtained:PRV-S^11-10,PRV-S^35-36,PRV-S^46-27.2 Biological characteristics of recombinant pseudorabies virus harboring porcine epidemic diarrhea virus S geneTo study the influence of insertion sites of PEDV S gene expression cassette into the non-coding region UL11 and UL10,UL35 and UL36,UL46 and UL27 of PRV genome,the growth kinetics of PRV recombinants were determined in vitro,The results showed that the growth characteristics of three recombinant viruses PRV-S^11-10、PRV-S^35-36、PRV-S^46-27 in vitro were similar to that of PRV^{ΔTK&g E-AH02LA},and reached the peak titers at 48 h after inoculation,the virus titers were 10^7.87 TCID₅₀/m L,10^8.27 TCID₅₀/m L and 10^8.05 TCID₅₀/m L,respectively,indicating that the insertion of foreign gene into these three sites had no significant effect on the growth characteristics of the recombinant strain in vitro.The virus titers of three recombinant viruses and the parent strain were significantly lower than the virulent PRV AH02LA strain at 6 h,36 h,48 h and 60 h post infection,indicating that deletion of TK&g E genes reduced the replication ability of the recombinant strain in vitro.The results of genetic stability test showed that the three recombinant viruses could stably express S gene after 20 passages in ST cells,and there was no deletion or site mutation of S gene proved by sequencing,indicating good genetic stability of the three recombinant viruses.The results of indirect immunofluorescence showed that the S protein could be stably expressed in ST cells infected with the three recombinant virus.The results of real-time PCR showed high transcription levels of S gene of all the three recombinant virus even though there were differences in these recombinants,among which PRV-S^11-10 showed the highest transcription level of S gene.In conclusion,the recombinant virus constructed by inserting PEDV S gene into the non-coding region of PRV genome between UL11 and UL10,UL35 and UL36,UL46 and UL27 could stably express PEDV S gene,and had no significant effect on the original biological characteristics of PRV.