Construction Of Recombinant Duck Plague Virus With Ul23 And US10 Gene Loss By Using CRISPR-Cas9 Technology

Duck plague is an acute,febrile,septic infection of poultry caused by the duck plague virus.The CRISPR-Cas9 system is a gene-editing technique,CRISPR-associated protein 9 could accurately cut target gene fragments.In this study,UL23 and US10 genes of DPV were used as targets.The left and right homology arms of UL23 and US10 were amplified by using specific primers,and the recombinant plasmids PUC19-UL23-EGFP and PUC19-US10-EGFP were constructed by using PUC19-EGFP,the linearization of UL23 and US10 were using as the target gene fragments UL23(AB)-EGFP and US10(AB)-EGFP by using the PCR method.furthermore,UL23 and US10 gRNA(unidirectional RNA)were designed and synthesized,and PX-330 Cas/gRNA(PX330-UL23-Cas9/gRNA plasmid and PX330-US10-Cas9/gRNA plasmid)were constructed by respectively using PX-330 plasmids.The corresponding linearized gene fragment and PX-330 Cas/gRNA plasmid were co-transfected into chicken embryo fibroblasts.According to the expression of green fluorescent protein and cytopathic conditions which are combined with plaque purification technique and PCR methods,finally rescued the missing UL23 and US10 gene to recombine duck plague virus?UL23 and ?US10.The experimental results laid a good material basis for the development of a new duck plague vaccine.