Construction Of Tk Gene Deletion Of PRV-XJ/?gE/gI-VP7.S Strain And Its Partial Biological Characteristic Analysis

Pseudorabies(PR)is an important infectious disease which threatens the pig industry.The outbreak of PR in China since 2011 was caused by the variant Pseudorabies Virus(PRV)strains.Considering the evolution of the strains,many researchers began to explore the development of new PRV vaccines.Porcine epidemic diarrhea virus(PEDV)and Porcine rotavirus(Po RV)infection in pigs can lead to substantial economic losses,especially variant PEDV strains since 2010.As a member of the Herpeviriade family,PRV is eligible for constructing recombinant subunit vaccines.Our laboratory successfully constructed a recombinant PRV strain expressing PEDV-S and PORV-VP7 gene(PRV-XJ?gE/gI-VP7.S)by homologous recombination technology based on PRV-XJ strain in 2018.As for the strain,PEDV-S(475-804 aa)and PORV-VP7(17-339 aa)were inserted at the deletion sites of gE and gI.Besides,a marker gene EGFP was also introduced.TK is an essential virulence gene of PRV,some previous studies have shown that the deletion of it can further enhance the immunogenicity of the virus.In order to construct effective vaccine candidates,this experiment intends to delete TK gene of PRV-XJ?gE/gI-VP7.S utilizing CRISPR/Cas9 technology.In addition,some biological characteristics of this strain,including genetic stability,proliferation characteristics,as well as animal safety were tested.1.Construction of Lentcrispr-g1 and Lentcrispr-g2 vectorsFirstly,the whole TK gene of PRV-XJ was amplified and sequenced,and the genetic information was submitted to Gen Bank database of NCBI.DNASTAR7.1 software was used to compare the homology of TK gene with those formerly isolated 28 strains.As a result,All the strains involved share the homology between 99.1%-100%.PRV-XJ extremely approximates which were isolated after 2011.On top of that,genetic evolution analysis reveals those Chinese strains locate in the same branch.Amino sequence analysis demonstrates that there are a few mutations in domestic strains.The CDS of TK gene was submitted to the website(http://crispr.mit.edu)and two pairs of gRNAs were chosen according to the ranking scores.Based on Lenticrispr-V2 vector,two CRISPR vectors were finally constructed,named Lentcrispr-g1 and Lentcrispr-g2 respectively.2.Construction of PRV-XJ?gE/gI/TK-VP7.S strainIn this study,I knocked out TK gene by means of transfection-infection based approach.BHK-21 cells in 12-well plates were co-transfected with Lentcrispr-g2 and PUC57-TK-RED homologous donor which was previously constructed and kept in our laboratory.10?L PRV-XJ?gE/gI-VP7.S was inoculated 24 h after transfection,and the virus was purified by infinite dilution method.550-960 bp of TK gene was ligated to Pcold-TF vector and then transfected into BL21 E.coli.The prokaryotic expression protein was purified by Ni-NTA column and immunized the SPF mice for three times.10 d past the third immunization,the blood sample was collected and used as the Primary antibody for Western-blot.Ultimately,both PCR and Western-blot confirmed that the virus was completely constructed,named PRV-XJ?gE/gI/TK-VP7.S.3.Partial biological characteristics of PRV-XJ?gE/gI/TK-VP7.S strainThe morphology of PRV-XJ?gE/gI/TK-VP7.S and PRV-XJ in PK-15 cells were observed by transmission scanning electron microscopy.The results showed that there was no discrepancy between both viruses,which had a diameter of 80 nm simultaneously.BHK-21 cells were used to multiply PRV-XJ?gE/gI/TK-VP7.S for 21 consecutive times,and PCR amplification demonstrated that the deletion of TK gene was stable.The one-step growth curve of PRV-XJ?gE/gI/TK-VP7.S resembles the parental strain PRV-XJ?gE/gI-VP7.S and PRV-XJ.However,the titer of PRV-XJ?gE/gI/TK-VP7.S was the lowest among them,but such difference gradually disappeared after the 48 h(P<0.05).In the meanwhile,the viral plaque of PRV-XJ?gE/gI/TK-VP7.S was the smallest.The safety test in mice showed that PRV-XJ?gE/gI-VP7.S has the LD₅₀<2×10³TCID₅₀,while PRV-XJ?gE/gI/TK-VP7.S shares the LD₅₀>10⁷TCID₅₀,so the safety of the strain was significantly improved after TK gene deletion.In addition,5×10⁶TCID₅₀dose of PRV-XJ?gE/gI/TK-VP7.S was safe for newborn piglets,weaned piglets,nursery piglets and pregnant sows.