Construction Of Two Recombinant Herpesviruses Of Turkey Expressing EGFP RHVT-US2/US10-EGFP By Using CRISPR/Cas9 Technology

This study intends to establish an vector flatform for HVT by the method of CRISPR/Cas9 technolgy to edit genes,and resucue recombinant HVT viruses which insert foreign gene within US2 or US10 genes.First,designed primers for g RNA accoding to the sequeces of US2 gene for HVT FC-126 strain,and acquired the aim fragment using the method of double chain annealing.This fragment inserted into the Bbs I digested px330 vector.The recombinat plasmid called px330-gRNA.Using PCR method,homologous arms A and B seperately located both sides of US2 gene and EGFP sequences encoding protein were amplified,and then fused into a whole fragemnt US2A-EGFP-US2 B.px330-gRNA and US2A-EGFP-US2 B were electrotransfected to chicken embryo fibroblasts.The recombinat viruse r HVT-US2-EGFP stably expressed EGFP was obtained by plaque purification.In the same way,the recombinat viruse rHVT-US10-EGFP were acquired.These results indicated that HVT recombinant expression vector systems were established,which were the basis for resucing recombant HVT viruses stably expressiong foreign proteins and applificating in future.