Cloning the Structure Genes and Expression the N Gene of Porcine Epidemic Diarrhea Virus DX
10.1007/s12250-009-2982-y
- Author:
Jianqiang LI
;
Jixing LIU
;
Xi LAN
;
Jie CHENG
;
Run WU
;
Zhongzi LOU
;
Xiangping YIN
;
Xuerui LI
;
Baoyu LI
;
Bin YANG
;
Zhiyong LI
- Publication Type:Journal Article
- Keywords:
Cloning;
Structure genes;
N gene;
Expression;
PEDV
- From:
Virologica Sinica
2009;24(3):179-186
- CountryChina
- Language:Chinese
-
Abstract:
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.
