[Objective] The aim was to construct a recombinant pseudorabies virus expressing canine distemper virus H gene and investigate its biological characters.[Method] H gene of canine distemper virus（CDV）strain Onderstep...[Objective] The aim was to construct a recombinant pseudorabies virus expressing canine distemper virus H gene and investigate its biological characters.[Method] H gene of canine distemper virus（CDV）strain Onderstepoort was produced by RT-PCR,inserted into pcDNA3.1（＋）vector to construct a expression cassette,which was then subcloned into transfer vector p8AA,prior to the insertion of LacZ expression cassette.The resulting new transfer vector was named as p8AAZH.Subsequently,p8AAZH was co-transfected with the genome of pseudorabies virus（PRV）Bartha-K61 into BHK-21 cells to enable gene recombination and virus package,and the virus solution was collected as cytopathic effect occurring.A series of procedures including blue plaque purification,PCR identification,observation under electron microscope and Western blot were carried out to screen the recombinant pseudorabies virus and identify the protein expression of target gene.Meanwhile,growth curve of the recombinant virus was determined in BHK-21 cells.[Result] The H gene had been inserted into the genome of Bartha-K61 strain,and RPRV-H was the same as Bartha-K61 in the one-step growth curve and cytopathic effect in BHK-21 cells.[Conclusion] The recombinant pseudorabies virus was constructed,and the insertion of H gene did not influence proliferation of recombinant virus,which laid a foundation for development of recombinant canine distemper virus vaccine.展开更多
In the present study, we examined the codon usage bias between pseudorabies virus （PRV） US1 gene and the USl-like genes of 20 reference alphaherpesviruses. Comparative analysis showed noticeable disparities of the s...In the present study, we examined the codon usage bias between pseudorabies virus （PRV） US1 gene and the USl-like genes of 20 reference alphaherpesviruses. Comparative analysis showed noticeable disparities of the synonymous codon usage bias in the 21 alphaherpesviruses, indicated by codon adaptation index, effective number of codons （ENc） and GC3s value. The codon usage pattern of PRV US1 gene was phylogenetically conserved and similar to that of the USl-like genes of the genus Varicellovirus of alphaherpesvirus, with a strong bias towards the codons with C and G at the third codon position. Cluster analysis of codon usage pattern of PRV US1 gene with its reference alphaherpesviruses demonstrated that the codon usage bias of USl-like genes of 21 alphaherpesviruses had a very close relation with their gene functions. ENc-plot revealed that the genetic heterogeneity in PRV US1 gene and the 20 reference alphaherpesviruses was constrained by G＋C content, as well as the gene length. In addition, comparison of codon preferences in the US1 gene of PRV with those ofE. coli, yeast and human revealed that there were 50 codons showing distinct usage differences between PRV and yeast, 49 between PRV and human, but 48 between PRV and E. coil Although there were slightly fewer differences in codon usages between E.coli and PRV, the difference is unlikely to be statistically significant, and experimental studies are necessary to establish the most suitable expression system for PRV US1. In conclusion, these results may improve our understanding of the evolution, pathogenesis and functional studies of PRV, as well as contributing to the area of herpesvirus research or even studies with other viruses.展开更多
文摘[Objective] The aim was to construct a recombinant pseudorabies virus expressing canine distemper virus H gene and investigate its biological characters.[Method] H gene of canine distemper virus（CDV）strain Onderstepoort was produced by RT-PCR,inserted into pcDNA3.1（＋）vector to construct a expression cassette,which was then subcloned into transfer vector p8AA,prior to the insertion of LacZ expression cassette.The resulting new transfer vector was named as p8AAZH.Subsequently,p8AAZH was co-transfected with the genome of pseudorabies virus（PRV）Bartha-K61 into BHK-21 cells to enable gene recombination and virus package,and the virus solution was collected as cytopathic effect occurring.A series of procedures including blue plaque purification,PCR identification,observation under electron microscope and Western blot were carried out to screen the recombinant pseudorabies virus and identify the protein expression of target gene.Meanwhile,growth curve of the recombinant virus was determined in BHK-21 cells.[Result] The H gene had been inserted into the genome of Bartha-K61 strain,and RPRV-H was the same as Bartha-K61 in the one-step growth curve and cytopathic effect in BHK-21 cells.[Conclusion] The recombinant pseudorabies virus was constructed,and the insertion of H gene did not influence proliferation of recombinant virus,which laid a foundation for development of recombinant canine distemper virus vaccine.
基金supported by grants from the Scientific Research Foundation for the Ph.D.,Guangzhou Medical University(2011C20)National Natural Science Foundation of China(31200120)+1 种基金Medical Scientific Research Foundation of Guangdong Province,China(B2012165)the Guangzhou city-level key disciplines and specialties of Immunology(B127007)
文摘In the present study, we examined the codon usage bias between pseudorabies virus （PRV） US1 gene and the USl-like genes of 20 reference alphaherpesviruses. Comparative analysis showed noticeable disparities of the synonymous codon usage bias in the 21 alphaherpesviruses, indicated by codon adaptation index, effective number of codons （ENc） and GC3s value. The codon usage pattern of PRV US1 gene was phylogenetically conserved and similar to that of the USl-like genes of the genus Varicellovirus of alphaherpesvirus, with a strong bias towards the codons with C and G at the third codon position. Cluster analysis of codon usage pattern of PRV US1 gene with its reference alphaherpesviruses demonstrated that the codon usage bias of USl-like genes of 21 alphaherpesviruses had a very close relation with their gene functions. ENc-plot revealed that the genetic heterogeneity in PRV US1 gene and the 20 reference alphaherpesviruses was constrained by G＋C content, as well as the gene length. In addition, comparison of codon preferences in the US1 gene of PRV with those ofE. coli, yeast and human revealed that there were 50 codons showing distinct usage differences between PRV and yeast, 49 between PRV and human, but 48 between PRV and E. coil Although there were slightly fewer differences in codon usages between E.coli and PRV, the difference is unlikely to be statistically significant, and experimental studies are necessary to establish the most suitable expression system for PRV US1. In conclusion, these results may improve our understanding of the evolution, pathogenesis and functional studies of PRV, as well as contributing to the area of herpesvirus research or even studies with other viruses.
