Cloning the Structure Genes and Expression the N Gene of Porcine Epidemic Diarrhea Virus DX  

作　　者：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 

机构地区：[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

出　　处：《Virologica Sinica》2009年第3期179-186,共8页中国病毒学（英文版）

基　　金：National Basic Research Program (2004CCA00500);National High-tech Development Research Program of China (2006AA02Z440)

摘　　要：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.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.

关 键 词：CLONING Structure genes N gene EXPRESSION PEDV 

分 类 号：Q785[生物学—分子生物学] S852.65[农业科学—基础兽医学]