Construction Of Artificial Chromosome And Gene Mutation Of Pseudorabies Strain LA2017

Pseudorabies is a severe animal infectious disease caused by Pseudorabies virus.The disease can cause death of piglets,recessive infection of finishing pigs,decline of boar fecundity,abortion or stillbirth of sows,and cause hμge economic losses to the global pig industry.Vaccine is the best way to prevent Pseudorabies,since 2011,there are still outbreaks of Pseudorabies in many pig farms using traditional Bartha-K61 vaccine.Therefore,it is urgent to construct and screen a new type of Pseudorabies Vaccine.1 Construction and identification of artificial chromosome of attenuated strain LA2017In 2012,AH02LA strain with PRV mutation was isolated and identified in our laboratory.After natural passage of F151 on chicken embryo fibroblasts,a attenuated virus with PRV passage was obtained and named LA2017.Previous studies have shown that the strain is safe and does not cause peer transmission in piglets and sows.Compared with Barthar-K61 vaccine strain,LA2017 group produced significantly higher levels of neutralizing antibodies 14 days after inoculation than Barthar-K61.In order to explore the Weakening Mechanism of LA2017 strain,we first established the reverse genetic operation platform of LA2017 strain,and successfully constructed the Bacterial artificial chromosome of LA2017 strain.After estriction enzyme digestion,p LA2017-BAC plasmid conformed to the whole genome characteristics of LA2017;after co-transfection of p LA2017-BAC plasmid and g C replaced gene fragment into CEF cells,the PRV LA2017-BAC and g C gene restorer PRV LA2017-Rec were rescued respectively by spot picking,screening and purification.The growth kinetics of AH02LA,LA2017,PRV LA2017-BAC and PRV LA2017-Rec strains were determined.The results showed that the growth kinetics of AH02LA strain was significantly different from that of the other three strains.The parent strain AH02LA grew rapidly and had high titer.The highest titer of AH02LA reached 10^8.33TCID₅₀/m L within 40～48h after inoculation,which was significantly higher than that of other strains（P=0.042）.However,the growth characteristics of LA2017 strain,PRV LA2017-BAC and PRV LA2017-Rec were basically the same（P=0.021）.The highest titer of LA2017 strain,PRV LA2017-BAC and PRV LA2017-Rec was 10^6.79,10^6.70 and 10^6.62TCID₅₀/m L respectively within 68～72 h after inocu Lation.Therefore,the p LA2017-BAC constructed is the Bacterial artificial chromosome of the whole genome of LA2017 strain.The in-depth study of p LA2017-BAC is conducive to reveal the internal mechanism of affecting the virulence and growth characteristics of PRV.2 Sequence analysis of the genome of porcine Pseudorabies virus LA2017The whole genome of pLA2017-BAC plasmid was sequenced by high-throμghput sequencing.According to the PRV mutant strain ZJ01 as the reference sequence,throμgh comparative analysis,the mutation sites and deletion sequences of the genome of the attenuated strain LA2017 were obtained;primers were designed,and the mutation sites were further verified by PCR amplification and sequencing using the DNA of the parent strain AH02LA and the sub-cultured strain LA2017 as templates.The results showed that LA2017 strain had three kinds of gene mutations compared with AH02LA strain,including deletion mutation,insertion mutation and allele mutation,which led to synonymous and non-synonymous amino acid mutations.Among them,the gene deletion sites were mainly concentrated in the coding region US7、US8、UL47（117（ED）⁺、G218A）、UL46（569E^-）、UL30（V686M）、UL32（R372H）、UL38（A58S）、UL26.5[A227S、229（APVQAAAPAAPAA PASAP）^-]、US4（R331H）.There was a deletion mutation of 665bp in the Non-coding regions between UL46 and UL27 genes in LA2017.Through this study,we can find that there are several gene mutations in the coding and non-coding regions of the attenuated LA2017 strain.The subsequent analysis of the function of each gene mutation site will help to reveal the new virulence genes of PRV epidemic variant strains,and provide a theoretical basis for the development of new safe and effective attenuated strain vaccine.