Construction Of BHV-1 GE Deletion Strain Based On CRISPR/Cas9 Technology And Preliminary Identification Of Its Biological Characteristics

Bovine(Bos taurus)herpes virus?(BHV-1)could cause infectious bovine rhinotracheitis(IBR)and result in economic losing in cattle raising industry.In recent years,the development of bovine nasal bronchitis vaccine has been carried out at home and abroad.For traditional vaccines,inactivated vaccines can not or rarely cause cellular immunity,and attenuated vaccines have toxic and resilience.For novel vaccines,subunit vaccines cannot replicate in vivo,and most DNA vaccines can only cause partial humoral immune responses.However,gene-deficient vaccines have immunological markers,and the differential diagnosis of wild-type infections and vaccine immunizations can be made using the missing genes,which has become the mainstream of new vaccine research and development.In order to construct gene deleted vaccines more efficiently,the present study combined CRISPR/Cas9 gene editing technology and homologous recombination technology to exchange the glycoprotein E(gE)gene of BHV-1 with green fluorescent protein gene(EGFP),named as BHV-1 gE~-/EGFP~+.Subsequently,the EGFP gene in BHV-1 gE~-/EGFP~+was directly removed by specific small guide RNA(sgRNA),and the gE deleted virus was named as BHV-1?gE.Transfection of pCas9-mCherry plasmid into MDBK,BTC,HEK293T and VERO cells by liposome method.By observing red fluorescence,flow cytometry for semi-quantitative analysis,and observation of different cells infected with BHV-1,The results showed that VERO cells not only had high transfection efficiency,but also were suitable for infection with viral BHV-1,so VERO cells were selected to edit the genome of BHV-1.The PCR identification and sequencing results of the recombinant virus BHV-1 gE~-/EGFP~+and BHV-1?gE plaque clones showed that BHV-1 gE~-/EGFP~+lacked 1134 bp at the 5'start of gE gene,and BHV-1?gE had Was built successfully.Biological characteristics analysis showed that BHV-1?gE had similar growth characteristics and plaque morphology when compared to the parental BHV-1.Although BHV-1 can be used as a living carrier to carry foreign antigens of cattle,the genetic recombination technology of BHV-1 has yet to be improved.The study of recombinant BHV-1 virus constructed by CRISPR/Cas9 technology has not been reported yet.The present study explored the BHV-1 virus genome editing with CRISPR/Cas9 system which could provide an efficient method for constructing BHV-1 gene deleted strains and providing a preliminary experimental basis for the future development of the IBR gene deletion vaccine.