key: cord-014932-web2tdef
authors: Li, Jian-qiang; Liu, Ji-xing; Lan, Xi; Cheng, Jie; Wu, Run; Lou, Zhong-Zi; Yin, Xiang-ping; Li, Xue-rui; Li, Bao-yu; Yang, Bin; Li, Zhi-yong
title: Cloning the structure genes and expression the N gene of porcine epidemic diarrhea virus DX
date: 2009-05-28
journal: Virol Sin
DOI: 10.1007/s12250-009-2982-y
sha: 
doc_id: 14932
cord_uid: web2tdef

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.

several subgenomic mRNAs for the production of structure and non-structure proteins (6, 18) . (1, 5) . The M and sM proteins are essential for viral envelope formation and release, the M protein also can stimulate the production of interferon (IFN) (1, 7) . The N protein participates in transcription of the viral genome, the formation of the viral core, and packaging of viral RNA. In the early stage of PEDV infection, the pig produces high levels of antibodies against the protein N. Since the N protein is highly conservative in the coronaviruses, it has a good response and immunogenicity, so it is the best candidate protein for early diagnosis reagents and vaccine development.

Although some nucleotides sequences of PEDV isolated from China has been reported, the data of DX structure genes are useful for furthering the study of the molecular biology of PEDV strains that are prevalent in China, especially in the north-west. In this study, the structure genes have been cloned and the N protein has been expressed.

The PEDV DX strain was collected from the feces of piglets suffering from severe diarrhea in Gansu north-west China. Total RNA was isolated from purified feces samples and extracted using a RNA extraction kit (Qiagene, Germany) following the manufacturer's instructions.

The RT-PCR amplifications were carried out using five primer sets (Table 1) and an RT-PCR amplification Kit (Toyobo, Japan). The products were ligated with the pMD18-T vector (TaKaRa) and transformed into the competent E. coli JM109. Positive clones were sequenced by the TaKaRa Biotechology (Dalian) Co.Ltd.

Phylogenetic analysis was performed for the amino acids sequence of PEEV DX strain structure genes and compared to the PEDV reference strains retrieved from GenBank. The sequence data were aligned using 

Two PCR primers of PEDV N, which contain specific restriction enzyme digestion sites depending on the multiple cloning sites contained in the expression vector pET30a were used. The sense primer 

The positive recombinant transformant was grown in LB media containing 100μg/mL Amp while shaking at 220 r/min at 37℃ and then induced with IPTG.

Cells were harvested by centrifugation at 12000 r/min for 1 min. Total cellular pellets were analyzed by 10% SDS-PAGE and Western blotting.

Using RT-PCR, two overlapping products of the S gene of approximately 4.2 kb were amplified ( Fig.1 

To analyze the phylogenetic relationships between DX and other PEDV strains isolated in various parts of the world, we constructed 3 neighbor-joining phylogenetic trees (Fig.2, Fig.3, Fig.4) 

The recombinant plasmid pET30-PN was digested with Ncol I and BamH I producing two fragments of 5 400bp (pET30a) and 1 400bp. PCR identification confirmed the presence of the 1 400bp fragment (Fig.6) .

To obtain the expressed recombinant protein, the time course expression of recombinant plasmid pET-PN was induced by IPTG with ranging from 0.5-1 mmol/L at 2, 3, 4, 5 and 6 h respectively. SDS-PAGE revealed that the best condition was the IPTG with 1 mmol/L at 6 h. The expressed proteins had a molecular weight of 55kDa as expected (Fig.7) . Western blot showed the The N protein is a RNA binding protein, it contains basic amino acids such as Arginine and lysine and shows a high degree of alkalinity, it is also the only phosphorylated structure protein in the coronavirus.

The Serine residue is a potential phosphorylation site

(2). TheDX N protein contained 37 Arginine, 33

lysine and 36 Serine. The N protein is the most highly expressed protein in coronavirus infected cells and is about 45kDa-60kDa (12) . The PEDV N protein is about 49kDa-58kDa in different expression systems

(2). In this study, an N protein of about 50kDa (the expressed vector was abou 5kDa) was obtained using the E. coli system, the result is consistent with other E.

coli expression system, although there are differences in the observed weights (16) . Western blot results showed the expressed N protein reacted with the antibodies, which indicated that the expressed N protein has the biological activity. Our results provide a basis for further development of a diagnosis method.

The production of recombinant infectious DI-particles of a

Sequence of the spike protein of the Porcine epidemic diarrhea virus

Characterization of the structural proteins of porcine epidemic diarrhea virus, strain CV777

Cloning and sequence analysis of the M gene of Porcine epidemic diarrhea virus LJB/03

Cloning and sequence analysis of the N gene of porcine epidemic diarrhea virus LJB/03

Serologic study of the occurrence of epizootic viral diarrhea in swine in Switzerland

Cloning and sequence analysis of the Korean strain of spike gene of Porcine epidemic diarrhea virus and expression of its neutralizing epitope in plants

Cloning and Sequence Analysis of the Nucleocapsid Gene of Porcine Epidemic Diarrhea Virus Chinju99

The molecular biology of coronaviruses

Erhebungen uber porcine coronaviren in osterreich II porcine epidemic diarrhea virus (PEDV) der schweine

Complete Genome Sequence of Transmissible Gastroenteritis Coronavirus PUR46-MAD Clone and Evolution of the Purdue Virus Cluster

Isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages

A novel internal open reading frame product expressed from a polycistronic of porcine edpidemic diarrhea virus may not contribute to virus attenuation

Disease of Swine

A cell culture vaccine against bovine ephemeral fever

Cloning and sequence analysis of the spike gene of Porcine epidemic diarrhea virus Chiju99

The nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein A1 in vitro and in vivo


