Construction And Biological Characterization Of Recombinant Feline Herpesvirus ? Lacking US3 Expression

Feline herpesvirus 1(FHV-1)is one of the Varicellovirus genus of the herpesvirus subfamily Alphaherpesvirinae,which can cause fever,ocular,nasal mucosa damage and obvious symptoms of upper respiratory tract infection.Cat is the mainly natural host of FHV-1.The mortality rate is up to 50% and the incidence rate is as high as 100%.US3 gene,encoding serine/threonine protein kinase,exists in all Alphaherpesvirinae.It is an important virulence gene and is highly conserved.This gene is mainly associated with the process of membrane fusion,cell rearrangement,anti-apoptosis,and enhancement of virus resistance.Previous studies about HSV and PRV suggested that viral titers were reduced when the US3 gene was knocked out,meanwhile the number and size of plaque decreased significantly with the increase of interferon.Firstly,the polyclonal antibody against FHV-1 US3 protein was prepared.In this study,the US3 gene was amplified by PCR and cloned into prokaryotic expression vector p ET-32 a to obtain the plasmid p ET32-US3,then ransformed into Escherichia coli BL21(DE3).Expression of His tagged US3 recombinant protein was induced by 1.0 mmol/L IPTG.The results of SDS-PAGE and Western blot showed that the recombinant protein was soluble and had good immunogenicity.Then,polyclonal antibody was prepared by immunization with purified protein.The titer of indirect ELISA was 1: above 10 000.The results of Western blot and IFA showed that the polyclonal antibody was able to react well with US3 protein,and provided the experimental material for the identification of r FHV-del US3.Secondly,according to the published FHV-1 C-27 gene sequence of Gen Bank,primers for amplifying the homogenous arms were designed.Both sides of the US3 gene were amplified to obtain the left homologous arm(L)and the right homologous arm(R)using FHV-1 VR-814 strain as the parent virus.The fragment L and R cloned into pc DNA3.1(+)vector in which CMV promoter was deleted.The expression box of CMV-EGFP was inserted between L and R to obtain the transfer vector pc DNA3.1-LR-EGFP.After the plasmid was transfected into F81 cells 12 h,the cell was inoculated with parental virus.US3 gene deletion virus was obtained by plaque screening and purification.Finally,the r FHV-del US3 with a US3 gene deletion was identified by PCR and Western blot,and the results showed that the US3-null mutant was successfully constructed.Finally,in order to clarify the biological characteristics of r FHV-del US3 including the genetic stability,the effect on the activation of IFN-beta promoter,the growth curve in vitro and the pathogenicity in mice were tested.The results showed that r FHV-del US3 can grow stably in CRFK cells.Visible green fluorescence from the tenth passage could be observed.One step growth curve and plaque formation test showed that the plaque size of r FHV-del US3 was reduced and the replication ability was decreased.The ability to inhibit the activation of IFN-beta promoter was significantly decreased.The results of pathogenicity test showed that the survival rate of the parent virus group was 75%,and the r FHV-del US3 group was 100%.Moreover,the level of virus DNA in lung tissue of r FHV-del US3 group was significantly lower than that of the parental virus group.These results indicated that the pathogenicity of r FHV-del US3 was decreased.FHV-1 US3 gene deleted mutant was constructed through homologous recombination technology and the recombinant virus exhibited well genetic stability and good growth.The analysis of biological characteristics showed that the growth ability,the inhibition of IFN-? promoter and the pathogenicity of mice were decreased.