To construct a recombinant Newcastle disease virus expressing the gD protein of infectious laryngotracheitis virus,the ectodomain of the gD gene was fused with signal peptide,transmembrane domain and cytoplasmic tail,and then strategically inserted between P and M gene of the infectious cDNA clone pLa Sota-VIIF/HN,in which the F and HN genes of La Sota strain have been swapped with the corresponding genes from genotype VII NDV strain.The resulted recombinant plasmid pLaSota-VIIF/HN-gD was co-transfected with the helper plasmid pCIneo-NP-P-L into BHK-21 cells to rescue the recombinant chimeric NDV La Sota strain express-ing the gD protein of ILTV,named rLaSota-VIIF/HN-gD.After 10 passages in 9-day-old SPF chicken embryonated eggs,the recombinant rLaSota-VIIF/HN-gD were identified by PCR,indirect immunofluorescence assay(IFA)and western blot.The PCR was conducted using cDNA reverse transcribed from RNA of 10th passaged recombinant virus,with primers designed to flank the insertion site of the ILTV gD gene.BHK-21 cells infected with the 10th passage recombinant virus were utilized for IFA,employing a polyclonal antibody against ILTV gD protein.Western blot analysis was performed on the supernatant from recombinant virus-infected chicken embryos allantoic fluid adsorbed by chicken red blood cells and purified recombinant NDV virions released from the absorbed chicken red blood cells.The pathogenicity to 9-day-old chicken embryos and the replication dynamics of the 10th passage of the rLaSota-VIIF/HN-gD were also investigated preliminarily.PCR results confirmed the stability of the gD gene within the recombinant virus.The IFA and western blot analyses indicated that the gD protein was not only correctly expressed by rLaSota-VIIF/HN-gD but also successfully incorporated into the recombinant NDV virion.The chicken embryo pathogenicity test and replication dynamics study demonstrated that the rLaSota-VIIF/HN-gD maintained the characteristics of low pathogenicity and robust replication capability,with a mean death time of 168 hours in chicken embryos and an intracerebral pathogenicity index of 0.20.The peak 50%embryo infectious dose(EID50)of rLaSota-VIIF/HN-gD was determined to be 10-8.66/100μL.The recombinant virus developed in this study will provide a promising candidate for bivalent live vaccine candidate against co-infection of ILTV and genotype VII NDV.
infectious laryngotracheitis virusrecombinant chimeric Newcastle disease virusgD proteinbivalent live vaccine
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