Wu YanTao

Modification of the full-length cDNA clone of Newcastle disease virus isolated from an outbreak in the goose

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.