猪流行性腹泻病毒ORF3基因的克隆及序列分析

Cloning and Sequence Analysis of Porcine Epidemic Diarrhea Virus ORF3 Gene

为研究2015年重庆市荣昌部分腹泻猪场是否有猪流行性腹泻病毒的感染与流行,对猪流行性腹泻病毒ORF3(Open reading flame 3)基因进行克隆及序列分析研究。通过从疑似流行性腹泻病毒感染的猪场采集小肠组织,采用RT-PCR方法对猪流行性腹泻病毒的ORF3基因进行克隆。结果表明,所获得的ORF3基因ORF长675bp,编码223个氨基酸。通过DNAMAN软件分析,表明本研究中克隆的ORF3基因与CV777标准株(AF353511)的ORF3基因相似性为96.45%,与中国的NJ(KJ642645)的相似性最高,为99.41%,与attenuated DR13疫苗株(EU054930)相似性90.52%,而与CV777疫苗株(GU372744)相似性仅79.07%。系统进化树分析表明,该基因与中国毒株、韩国毒株以及美国分离株USA/Illinois 201/2014(KR265795)处于同一进化分支上,而attenuated DR13疫苗株、CV777 truncated疫苗株和欧洲的CV777标准株处于另一个进化分支上,说明该基因与中国毒株、韩国毒株的遗传距离最近。通过B细胞抗原表位预测分析,发现其氨基酸第65~68、114~116、137~140和150~154区域可能有潜在的优势抗原表位。成功克隆了猪流行性腹泻病毒ORF3基因,说明2015年重庆市荣昌部分腹泻猪场存在猪流行性腹泻病毒的感染。

To study whether some of pig farms with diarrhea was infected by porcine epidemic diarrhea virus in Rongchang of Chongqing,we cloned and analyzed sequence of the open reading flame 3（ORF3） gene of porcine epidemic diarrhea virus（PEDV） in this study.The small intestine tissue from piglets suffering from a severe diarrhea was collected.The PEDV ORF3 gene was amplified by using reverse transcription—polymerase chain reaction（RT-PCR）.The results showed that the amplified PEDV ORF3 gene contained 675 bp nucleotides sequence and encoded 223 amino acids.The similarity of theORF3 gene between the cloned gene and the CV777 standard strain（AF353511） was 96.45%.The highest similarity also showed that it was 99.41%from the China NJCKJ642645） by DNAMAN software analysis.The ORF3 Gene shared homology at 90.52%with the DR13 vaccine strain（EU054930）,while CV777 vaccine strain shared the lowest nucleotide homology at 79.07%.The phylogenetic analysis showed that the cloning gene and China strain,South Korea and the United States USA/Illinois strains isolated from 201/2014（KR265795） were in the same evolutionary branch,which they had a close phylogenetic ralationship.While attenuated DR13 and CV777 truncated vaccine strains and the European CV777 standard strain was in another evolutionary branch.The results showed that the amino acid 65 — 68,114—116,137 — 140 and 150 —154 regions may have potential advantage of epitopes by B cell epitope prediction analysis.We cloned porcine epidemic diarrhea virus ORF3 gene and the studies proved that porcine epidemic diarrhea virus was infected in part of pig farms with diarrhea in Rongchang of Chongqing in 2015.