Isolation And Identification Of Epidemic Variation JL1 Strain Of Porcine Pseudorabies Virus And Construction Of Transfer Vector For GE Gene Deletion

In recent years,the porcine pseudorabies has spread widely in many Bartha-K61 vaccine immunized pig farms in China.Related studies indicate that the outbreak of the disease is mainly caused by a variant of pseudorabies virus.The majority of pig farms was attacked by PRV infection.The rapid spread of the disease has seriously endangered the development of the pig industry in China and aroused much concern and research.In this study,virus isolation and identification of the tissue samples from a suspected PR farm in Jilin Province were collected,isolated PRV JL1 strain.The genetic variation of the major genes of the JL1 strain was analyzed to further understand the genetic variation of PRV.At the same time,the JL1 strain was used as the parent strain to construct the gE gene-deleted transfer vector,in order to lay the foundation for the subsequent development of gE gene-deleted strain.The research was divided into the following three parts:1.Isolation and identification of PRV JL1 strainTissue samples from a suspected pig pseudorabies pig farm in Jilin province were collected.After ground treatment,the suspected pseudorabies inoculated ST cells for virus isolation.The cells inoculated with the spleen supernatant showed obvious cell aggregation and fusion lesions after 24 h.Using PCR,it was found that the isolated virus was porcine pseudorabies virus and named JL1 strain.The results of animal backpaggage tests showed that the isolated strain had a strong pathogenicity to 2-month-old piglets.2.Genetic variation analysis of major genes of PRV JL1 strainSpecific primers were designed and synthesized,TK,g I,g D,gE and g B genes of JL1 strain were cloned,sequenced and analyzed with domestic and foreign strains from Genbank.The nucleotide homology of TK,g D,g I,gE and g B genes of JL1 strain with reference strains was 99.4%~100%,95.5%~99.9%,98.3%~99.9%,97.6%~99.9%,and 98.1%~99.9%,respectively.Amino acid homology was 98.8%~99.7%,92.4%~99.5%,96.5%~99.5%,95.5%~99.5%,and 96.4%~99.6%,respectively.The nucleotide and amino acid homology analysis results of each gene showed that the JL1 strain had high homology with the novel epidemic strains isolated in China,but had low homology with foreign classical strains.Amino acid sequence alignment results showed that there were two amino acids at position 48(D)and496(D)in gE of JL1 strain,which was consistent with that of the epidemic variant strains.At the same time,there are different numbers of amino acid insertion and deletion in g D,g I and g B gene,respectively.The results indicated that the deletion and variation of JL1 strain was the same as that of the domestic novel epidemic variation JS-2012 strain.Phylogenetic analysis results showed that PRV JL1 strain was closely related to the popular variant strains isolated in China in recent years,and differed from the classical strains in domestic-foreign country.The above results indicated that PRV JL1 strain belonged to a novel epidemic variant strain isolated in China in recent years.3.Construction of gE gene-deleted transfer vector of PRV JL1 straingE gene homologous arm,gEL and gER,and EGFP gene were amplified,restriction enzyme digested,ligased and cloned.gEL,EGFP and gER fragments were connected with the p Bluescript II SK expression vector,in order to construct PRV gE gene-deleted transfer vector p SK-gELR-EGFP.By enzyme digestion and sequencing,the transfer vector was successfully constructed.In addition,Fu GENE HD transfection reagent was used to transfect the vector into PK-15 cells,and the green fluorescence could be observed in transfected cells by inverted fluorescence microscope.These results indicated that the gE gene-deleted transfer vector was constructed and obtained,and the transfer vector can be expressed in PK-15 cells.In this study,the genetic evolution and molecular characteristics of the isolated PRV epidemic variation JL1 strain showed that the strain is a PRV epidemic variant strain.There was a large genetic difference between the JL1 strain and the foreign strains,especially the Bartha strain,which provided a theoretical basis for further explanation of the reasons for the sudden outbreak PR in China's Bartha vaccine-immunized farms.At the same time,the successful construction of the gE gene-deleted transfer vector with a screening marker EGFP.The vector laid the foundation for the development of gE gene-deleted vaccine strain and novel vaccine.