Cloning the structure genes and expression the N gene of porcine epidemic diarrhea virus DX

• DOI: 10.1007/s12250-009-2982-y
• Publication Date: 2009-05-27T06:38:58Z
• Authors: Jian-qiang Li; Ji-xing Liu; Xi Lan; Jie Cheng; Run Wu; Zhong-Zi Lou; Xiang-ping Yin; Xue-rui Li; Bao-yu Li; Bin Yang; Zhi-yong Li
• Keywords: 0301 basic medicine; 03 medical and health sciences; Article; 3. Good health

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.