伪狂犬病病毒gI/gE/TK基因缺失疫苗株的构建及免疫效力评价OA

This study aimed to rapidly construct a thymidine kinase(TK)-deleted pseudorabies virus(PRV)using CRISPR/Cas9 technology.Using JS-2012-ΔgI/gE as the parental virus,two single-guide RNAs(sgRNAs)targeting the TK gene were designed and co-transfected with the viral genome into BHK-21 cells.The recombinant virus PRV JS-2012-ΔgI/gE/TK was obtained via plaque purification,PCR screen-ing and sequencing identification.Subsequently,in vitrobiological characterization,in vivo safety tests and efficacy tests were conducted.Biological characterization revealed that the recombinant virus had consistent in vitro growth characteristics and plaque morphology with the parental virus,lost TK gene activity,and exhibited good genetic stability.Animal test results showed that no mortal-ity occurred in mice within 8 days after inoculation with 1×106 TCID50 of the recombinant virus;piglets inoculated with 1×106 TCID50 of the strain remained safe within 14 days and could be effective-ly protected against challenge with 1×105 TCID50 of virulent PRV.This study provides a theoretical basis for the development of safer pseudorabies vaccines and disease eradication.