Isolation And Identification Of PRV Mutant GZYY2015 And Construction Of GE Gene Deletion Transfer Vector

Pseudo rabies?PR?caused by pseudorabies virus?PRV?is a second-class animal disease that is strictly controlled and extinguished in China.There is no specific drug at present,and vaccination is still an effective measure to prevent the disease.The vaccine manufactured by Bartha-K61 strain,which naturally lacks the gE gene,has been widely applied to pig farms,and the effect is stable and good,which has made great contributions to the prevention and control of PR.Until the end of 2011,the pigs immunized with the classic vaccine strain Bartha-K61 again broke the PR epidemic,indicating that the classic vaccine strain Bartha-K61 can no longer provide complete immune protection for pigs.The sow reproductive failure and the mortality rate of newborn piglets are increasing,and the positive detection rate of PRV gE antibodies is also rising,which has brought huge economic losses to the pig industry.In the practice of pig production in Guizhou Province,we also often encounter PR disease in pigs that have been immunized with Bartha-K61 strain.In order to understand the PRV strain of such diseases,in order to better solve the PR problem in pig production,this study used a classic virus isolation method to isolate a PRV variant strain from clinical samples?GZYY2015?.Based on the gene sequence of this strain,the gE gene deletion transfer vector was constructed,which laid a foundation for the study of the PRV mutant gE gene deletion vaccine.1?Isolation and identification of PRV variant strain GZYY2015This study took a laboratory-diagnosed PRV-positive tissue sample from a sputum in the Yunyan area of Guiyang in 2015.After homogenization,freeze-thaw and sterile filtration,the filtrate was inoculated to a single layer of dense Vero cells were passaged to isolate PRV strains and some of their biological characteristics were identified.The experimental results show that after blind transmission to the 6th generation,PRV can form a stable CPE phenomenon on Vero cells characterized by cell swelling and rounding,aggregation and formation,and formation of vacuoles.Under electron microscope,the inclusion bodies in the nucleus of the virion showed a typical lattice arrangement.The negatively infected virions were round,and the icosahedral nucleocapsid structure was obvious,with typical morphological characteristics of PRV virions.The TCID₅₀0 was determined to be 10^-9.13TCID₅₀/0.1mL.The one-step growth curve showed that the virus was in a latent period within 12 h.The virus titer increased from 12 h to 48 h,and the virus titer became stable after 48 h.The strain was inoculated into rabbits.On the third day,the body temperature rose to41?,and itching symptoms appeared.The rabbit scratched the bite injection site,causing hair loss and bleeding.The body temperature dropped suddenly before death,and died 3 days after injection.They are all in line with the clinical features caused by pseudorabies virus infection,and they are named PRV GZYY2015 strain.In this study,TK,gE,gD and gC genes were cloned by T.The cloning plasmid and gB gene PCR products were sent to Shanghai Biotech for sequencing.The results showed that the size and ORF of each gene were 1535 bp?963 bp?and 1752 bp?1740bp?,1273 bp?1209 bp?,1651 bp?1464 bp?,and 2784 bp?2745 bp?.The sequence similarity analysis and genetic evolution tree were carried out respectively.The results showed that the TK,gE,gC and gB genes were all on the same evolutionary branch as the PRV variant epidemic strain,and had high similarity,while the gD gene sequence and Ea gene.The strains are identical and are located on the same evolutionary branch as the classical strains.These five genes are not in the same evolutionary branch as the foreign PRV strains.By analyzing the amino acid sequences of each gene,PRV GZYY2015 gE gene has an insertion of aspartic acid?D?at positions 48 and 496.It is consistent with the characteristics of PRV variant molecules reported by Zhao Hongyuan et al.Compared with the domestic mutant strain,the gC gene differs only from the JS strain at position303,and the H?histidine?mutation is R?arginine?.The gB gene is identical to the domestic variant strain.The amino acids 278 and 279 of the gD gene were inserted into S?serine?and P?valine?,and the variation of these two sites was different from that of the PRV mutant strain.The deletion of the amino acid at position 166 of the TK gene is also different from the PRV strain at home and abroad.In summary,the PRV GZYY2015 strain is in line with the characteristics of the PRV variant epidemic strain.This study enriches the PRV virus database in Guizhou Province and provides a scientific basis for the development and utilization of PRV-related variant strains.2?PRV GZYY2015 variant gE gene deletion transfer vector constructionPrimers were designed to amplify the homologous arms LA and RA of the upstream and downstream of PRV GZYY2015 mutant gE,and the green fluorescent protein EGFP-C1,promoter and partial multiple cloning sites were amplified from the primers designed from plasmid pEGFP-C1.First,the amplified gE left homologous arm LA fragment was digested,and then cloned into pcDNA3.1 eukaryotic expression vector to construct pcDNA3.1-LA plasmid.Then,the amplified gE right homologous arm RA fragment was digested,and then cloned into the pcDNA3.1-LA plasmid vector to construct a pcDNA3.1-LA-RA plasmid.Finally,the amplified EGFP-C1fragment was digested and ligated into the pcDNA3.1-LA-RA plasmid vector to construct a pcDNA3.1-LA-EGFP-RA plasmid.The relevant cloning plasmids were identified by enzyme digestion and sequenced.The sequencing results showed that the cloned LA-EGFP-RA fragment was 3571 bp in size,with Nhe ? and Xba ? restriction sites at both ends,and 835 bp to 2609 bp in the middle as EGFP sequence.Both ends were BamH ? restriction sites,sequencing.The results are in line with expectations.The successful construction of PRV gE deletion transfer vector laid the foundation for the development of PRV deletion vaccine for PRV mutant strain.