| Pseudorabies virus is a double-stranded DNA virus belonging to the herpesvirus family and the alpha-herpesvirus subfamily.It has a total length of about 150 kb and a GC content of 73%.Porcine pseudorabies is a disease caused by the pseudorabies virus of pigs,which is characterized by neurological symptoms and reproductive disorders,which seriously endangers the sustainable and healthy development of the pig industry in China.It is widespread in large-scale pig farms in various provinces of China.According to the report of Tongwu in 2013,since September 2011,PR diseases have occurred in various provinces of China,and pig farms with immune PRV vaccines still occur,and the morbidity and mortality have increased significantly.The results of isolation and identification of strains have shown that the disease has caused a large-scale outbreak.The pathogen is a variant of the pseudorabies virus.A similar PR disease has occurred in some farms in Guizhou province that have been immunized with the PRV Bartha-K61 vaccine strain.In 2016,a large-scale breeding farm in Danzhai County,Guizhou Province,in the case of immunized PRV Bartha-K61 vaccine,some pigs have abortion,stillbirth and mummified fetus,and then diagnosed as PRV wild poison by laboratory diagnosis.To.In order to understand the situation of PRV strain in this case,Vero cells were isolated and identified in this study.It was confirmed that the isolate strain was a PRV mutant strain(GZDZ2016),and TK was constructed based on the gene sequence of the strain.The gene deletion transfer vector laid the foundation for the study of the PRV mutant TK gene deletion vaccine.1.Isolation and Identification of PRV Mutant GZDZ2016This study took a sample of aborted fetal tissue diagnosed by PRV-positive pigs in a pig farm in Danzhai County in 2016.After serial treatment,it was inoculated into Vero cells for passage and separation,and the morphology of the isolated PRV was studied by electron microscopy.Observation,virus titer,one-step growth curve and partial gene sequence were measured.The results of isolation and identificationshowed that CPE began to be produced when it was blindly transmitted to the 5th generation,and it showed stable CPE when it was 7th generation.It showed typical CPE such as rounding,shedding,and pulling net.It can be seen in vesicles under transmission electron microscope.Mature virions with approximately circular shape in the nucleus.Virus inclusion bodies with lattice-like arrangement in the nucleus and hollow and solid non-enveloped virions;the titer of isolated virus on Vero cells For the 10-8.12/0.1mL,one-step growth curve showed that the virus titer was in the incubation period within 12 h,and it increased from 12 h to 56 h,and reached the maximum at 60 h;the isolated strain was inoculated In rabbits,symptoms of itching and hyperactivity appeared after 2 days,and died after 3 days.The 11 th generation cell culture medium was used for amplification of gD partial gene,and the sequencing results were analyzed.The PCR amplification products were sequenced and proved to be Partial sequence of the PRV gD gene.The results showed that a strain of pseudorabies virus Guizhou strain was successfully isolated and named as PRV GZDZ2016 strain.In this experiment,the gB,gC,gD,gE and TK genes of the isolates were amplified by PCR,and the expected sizes were: 2784 bp,1651 bp,1273 bp,1752 bp,1535 bp,and The gC,gD,gE and TK genes were cloned and sequenced,and the sequencing results were compared with the domestic and foreign strains' gene sequences and sequence similarity analysis.The phylogenetic tree was drawn by the deduced amino acid sequence.The results of sequence similarity analysis indicated that the main gene of the isolate had higher similarity with the mutant strain,but the similarity with the foreign strain was lower.Sequence alignment showed that the isolates gB,gC,gE and TK proteins had different numbers of amino acid variations,and the 48 th and 496 th gE proteins had aspartate(D)insertion,the 236 th Leucine(L)changes to proline(P);the 6th position of TK protein changes from isoleucine(I)to alanine(A),and position 157 changes from aspartic acid(D)Glycine(G);g-protein at position 81 changed from histidine(V)to alanine(A);gB protein and foreign strains have significant amino acid differences at 55,70,72-73;gD There was no amino acid difference between the protein and the mutant strains such as HeN1,TJ,and JS2012 strains isolated after 2012.The results of genetic evolution analysis showed that the isolate was closely related to the mutant strains isolated in recent years in China,and it was far away from the classical strains isolated at home and abroad.The above results indicate that the isolate belongs to a variant strain popular in China in recent years and is named as PRV variant strain GZDZ2016.2.Construction of PRK variant GZDZ2016 TK gene deletion transfer vectorThe specific primers were designed to amplify the TKL and TKR of the upstream and downstream homologous arms of the GKDZ2016 TK gene,and the amplification sizes were 1066 bp and 906 bp,respectively.The specific primers were designed to amplify the reporter gene LacZ from the pCMV-beta plasmid.The increase was 4062bp;the amplified products of TKL and TKR were ligated into pMD-19 T vector by T-cloning,and the HindIII-EcoRI fragment of pMD-TKR-19 T was ligated into pcDNA3.1 eukaryotic expression vector to construct pcDNA3.1-The TKR plasmid was 6334 bp in size and was positive by restriction enzyme digestion and plasmid PCR.The results showed that the construction was successful.The NheI-HindIII fragment of pMD-TKR-19 T was ligated into the pcDNA3.1-TKR plasmid vector to construct pcDNA3.1-TKL.-TKR plasmid,size 7400 bp,double enzyme digestion and plasmid PCR positive,indicating successful construction;finally,the reporter gene LacZ was double-digested and ligated into pcDNA3.1-TKL-TKR plasmid vector to construct pcDNA3.1-TKL The-LacZ-TKR plasmid,11462 bp in size,was confirmed to be successfully constructed by double digestion and sequencing. |
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