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Virologica Sinica, 24 (3) : 179-186, 2009
Research Article
Cloning the Structure Genes and Expression the N Gene of Porcine Epidemic Diarrhea Virus DX
1. College of Veterinary Medicine, Gansu Agricultural University , Lanzhou 730070, China
2. Key laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046,China
The structure genes spike (S), nucleocapsid (N), membrane (M), small membrane (sM) of a porcine epidemic diarrhea virus (PEDV) strain DX isolated in Gansu province, North-west of China, were cloned, sequenced and compared with published sequences of PEDV strains. The nucleotide sequences encoding the entire S, sM,M and N genes open reading frame (ORF) of DX were 4 152, 231,681 and 1 326 bases long respectively. There were transcription regulatory sequences (TRSs) upstream of the initiator ATG of the S, N and M genes. The amino acids sequences of S, M and N contained 30, 3 and 7 potential asparagine (N)-linked glycosylation sites. Homologous analysis and phylogenetic trees showed that DX had the closest relationship with strains LJB/06, JS-2004-2Z and CH/HLJH/06 that were also isolated from China and indicated the prevalence of some PEDV isolates in China were widespread since the JS-2004-2Z strain originated from the south of the China,and LJB/06 and CH/HLJH/06 were isolated from northeast China. The N gene was cloned using two primers which contained Nco I and BamH I restriction enzyme sites and subcloned into expression vector pET30a. The recombinant plasmid was then transformed into E.coli Rossta. SDS-PAGE showed there was a protein of about 55kDa as expected and Western blot indicated the N protein had biological activity.
Key Words: null
  Published online: 5 Jun 2009
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