Cloning and Construction of Eukaryotic Expression Vector for L Gene of Newcastle Disease Virus TL1 Strain

Objective To clone the L gene of Newcastle disease virus(NDV)TL1 strain,analyze its sequence and construct its eukaryotic expression vector.Methods Design a pair of primers according to the NDV L gene sequence reported in GenBank and amplify L gene from the NDV TL1 strain isolated in Inner Mongolia,China by RT-PCR.Purify the amplified gene and clone into pGEM-T easy vector,then subclone to eukaryotic expression vector pCI-neo.Identify the constructed recombinant plasmid by restriction analysis,PCR and sequencing.Results The amplified L gene,at a full-length of 6 704 bp and a ORF length of 6 615 bp,encoded 2 204 amino acids.The nucleotide and amino acid sequences at encoding region of L gene were compared with those of 12 reference strains in GenBank,and the result showed high homologies of TL1 strain to the NA-1,ZJ1 and SF02 strains of NDV from goose origin.ConclusionThe eukaryotic expression vector for L gene of NDV TL1 strain was successfully constructed,which laid a foundation of study on reverse genetics and functional genome of TL1 strain.