Preliminary Evaluation Of The Potential Of Duck Plague Virus UL6 Protein Nuclear Localization Signal Mutant Vaccine

Duck plague virus(DPV)is a member of the alpha herpesvirus subfamily.In the alpha herpesvirus subfamily,the UL6 protein generate functions in the nucleus,but the issue of how the DPV UL6 protein enters the nucleus has not been clarified.In this paper,through the construction of recombinant viruses and indirect immunofluorescence and other experimental methods,it was found that the UL6protein is located in the nucleus,which provides a theoretical basis for further elucidating the nuclear mechanism of the DPV UL6 protein targeting anti-DPV vaccine.This article focuses on the above purposes,the main research content and the results obtained are as follows:1.Identification of DPV UL6 protein nuclear localization signal(NLS)Recombinant virus DPV-UL6-Flag infected duck embryo fibroblast(DEF)cells for 24 h,UL6 protein was mainly located in the nucleus;eukaryotic expression plasmid p CAGGS-UL6 was transiently transfected into DEF and DF-1 cells,and UL6protein was located in the nucleus.Bioinformatics analysis showed that amino acids 126-157 and 732-746 were the double-type nuclear localization signal(bNLS)and the single-type nuclear localization signal(mNLS)of the UL6 protein;the corresponding UL6 protein mutants were constructed for the twoNLS segments.Indirect immunofluorescence found that UL6_126-790,UL6_158-790,UL6_?126-157,UL6_1-731and UL6_?732-746mutants were all located in the cytoplasm.This result shows that bNLS and mNLS have an important influence on the nuclear entry of UL6 protein,and there are sequences at the N-terminus of UL6 that have an important influence on the nuclear entry of UL6protein.After examining the N-terminal sequence of UL6,it was found that the UL6_111-790and UL6_?59-110mutants were mainly located in the cytoplasm,indicating that the amino acids 59-110 had an important effect on the nuclear entry of duck plague virus UL6 protein.2.Function detection of DPV UL6 proteinNLSThe amino acids 59-110,bNLS and mNLS of the UL6 protein were fused and expressed with the exogenous proteins EGFP and EGFP-?-Gal,respectively,to detect whether the three have nuclear localization signal ability.It was found that neither the amino acids 59-110 nor bNLS had the ability to carry foreign proteins into the nucleus through the indirect immunofluorescence.Only when the fusion expressed mNLS,the EGFP protein had obvious intranuclear aggregation.When mNLS and EGFP-?-Gal fusion showed nucleocytoplasmic states,indicating that mNLS has a weak nuclear localization signal ability.To further verify the function of the 59-110 amino acids,bNLS and mNLS nuclear localization signals,we conducted a synergistic test on the above three.It was found that,in the case of fusion expression of bNLS and mNLS,most of the EGFP protein was localized in the nucleus and appeared to be completely localized in the nucleus;the EGFP-?-Gal protein showed a nucleocytoplasm distribution,indicating that between bNLS and mNLS It has a synergistic effect,and amino acids 59-110 do not have the ability of nuclear localization signal.3.Identification of key amino acids inNLS of DPV UL6 proteinIn this experiment,the key amino 131R,142R,152K,737K,738R,and 740R in the UL6 protein mNLS and bNLS were mutated to alanine(A).It was found that131R and 152K had no effect on the nuclear entry of UL6 protein,142R and 737R had a certain effect on the nuclear entry of UL6 protein,and 738R and 740R had an important effect on the nuclear entry of UL6 protein.4.Preliminary evaluation of the potential of DPV-UL6_R142Amutant vaccineIn this paper,an infectious clone DPV-UL6_R142Awas constructed by Red recombination method.DPV-UL6_R142Aand DPV-BAC were inoculated into the allantois cavity of 10-day-old duck embryos,and observations were made from three aspects:lethality,changes in necropsy,and changes in virus copy number in duck embryos.In the experiment of detecting the survival rate of duck embryos,the duck embryos in the DPV-UL6_R142Agroup died later than the DPV-BAC group.The pathogenic ability of DPV-UL6_R142Aon duck embryos is similar to that of parent strain DPV-BAC.When detecting the replication of the virus in duck embryos,it was found that the proliferation time of the DPV-UL6_R142Amutant was later than that of the parent strain DPV-BAC,but the two eventually reached a similar replication platform period.This shows that the DPV-UL6_R142Amutant strain has similar pathogenicity and replication ability to duck embryos as parent strain.At the same time,the DPV-UL6_R142Amutant cannot replicate in the ducklage,so the DPV-UL6_R142Amutant does not have the potential to be a vaccine candidate.To sum up:Duck plague virus UL6 protein is mainly located in the nucleus when infected with virus and transiently expressed.The amino acids 59-110 of the UL6protein,bNLS,and mNLS all have an effect on the nuclear entry of the gate protein,but only mNLS has weak nuclear localization ability.There is a synergy between the UL6 protein bNLS and mNLS,which can jointly promote the protein into the nucleus.Among them,the 738R and 740R in mNLS are the key amino acids for UL6 protein to enter the nucleus.Although DPV-UL6_R142Amutant strain lags behind DPV-BAC in lethality of duck embryo and replication ability in duck embryo,it finally achieves lethality and replication ability similar to wild strain.At the same time,the DPV-UL6_R142Amutant cannot replicate in the ducklage,indicating that DPV-UL6_R142Amutant strain does not have the potential to become a vaccine.