A 6.5-kb specific fragment containing the T7 promoter and the transcription vector was excised from the full-length cDNA clone of the Newcastle disease virus(NDV)strain ZJI of goose origin,and thereafter it was self-l...A 6.5-kb specific fragment containing the T7 promoter and the transcription vector was excised from the full-length cDNA clone of the Newcastle disease virus(NDV)strain ZJI of goose origin,and thereafter it was self-ligated to form a high quality plasmid for mutagenesis.Site-directed mutagenesis was used for inserting three additional G nucleotides(nts)into the region between the T7 promoter and the leader sequence of the NDV genome.RT-PCR was employed to amplify the F/HN gene fragments,and then they were ligated by the shared restriction enzyme BsmBI.Finally,the corresponding fragment in the mutant full-length cDNA was substituted with the new one.The sequencing results showed that the three additional G nts were successfully inserted and the mutant nts in the full-length cDNA were corrected.This study lays a good foundation for research on the reverse genetics of NDV strain ZJI.展开更多
Six isolates of Newcastle disease virus were derived from south and east China regions during the disease outbreaks called "avian paramyxovirus infection of geese" or "geese paramyxovirus infection"...Six isolates of Newcastle disease virus were derived from south and east China regions during the disease outbreaks called "avian paramyxovirus infection of geese" or "geese paramyxovirus infection",and partial sequence analysis of hemagglutinin-neuraminidase(HN) gene was carried out to identify the genetic characteristics of these goose isolatesA 1905 nucleotide portion of HN gene of each of the 6 isolates was sequenced,the results revealed that the coding region of their HN genes are all 1716 nucleotides in length,which can encode 571 amino acid residues aloneThe coding region is followed by a noncoding sequence of 189 nucleotidesThough they diverged only 08%-37% from each other in the nucleotide sequences of coding region,they differed by 175%-179% to F48E8, a standard challenge strain of chicken originCysteine residues are well conserved throughout the amino acid sequences,while the number of the potential glycosylation sites varys from 4 to 6Residue positions Thr 48,His 54,Ser 77,Ala 266,His 340 and Lys 384 are also highly conserved in the 6 goose isolatesThe corresponding residues in other NDV strains are commonly Met 48,Ser 54,Asn 77,Ile 266,Tyr 340 and Glu 384However,the sequences of receptor-binding related regions show no difference to the 14 reference strains from domestic or展开更多
基金This work was supported by the National Natural Science Foundation of China(No.39893290).
文摘A 6.5-kb specific fragment containing the T7 promoter and the transcription vector was excised from the full-length cDNA clone of the Newcastle disease virus(NDV)strain ZJI of goose origin,and thereafter it was self-ligated to form a high quality plasmid for mutagenesis.Site-directed mutagenesis was used for inserting three additional G nucleotides(nts)into the region between the T7 promoter and the leader sequence of the NDV genome.RT-PCR was employed to amplify the F/HN gene fragments,and then they were ligated by the shared restriction enzyme BsmBI.Finally,the corresponding fragment in the mutant full-length cDNA was substituted with the new one.The sequencing results showed that the three additional G nts were successfully inserted and the mutant nts in the full-length cDNA were corrected.This study lays a good foundation for research on the reverse genetics of NDV strain ZJI.
文摘Six isolates of Newcastle disease virus were derived from south and east China regions during the disease outbreaks called "avian paramyxovirus infection of geese" or "geese paramyxovirus infection",and partial sequence analysis of hemagglutinin-neuraminidase(HN) gene was carried out to identify the genetic characteristics of these goose isolatesA 1905 nucleotide portion of HN gene of each of the 6 isolates was sequenced,the results revealed that the coding region of their HN genes are all 1716 nucleotides in length,which can encode 571 amino acid residues aloneThe coding region is followed by a noncoding sequence of 189 nucleotidesThough they diverged only 08%-37% from each other in the nucleotide sequences of coding region,they differed by 175%-179% to F48E8, a standard challenge strain of chicken originCysteine residues are well conserved throughout the amino acid sequences,while the number of the potential glycosylation sites varys from 4 to 6Residue positions Thr 48,His 54,Ser 77,Ala 266,His 340 and Lys 384 are also highly conserved in the 6 goose isolatesThe corresponding residues in other NDV strains are commonly Met 48,Ser 54,Asn 77,Ile 266,Tyr 340 and Glu 384However,the sequences of receptor-binding related regions show no difference to the 14 reference strains from domestic or
