猪流行性腹泻病毒FJ-11A株的分离与ORF3基因序列分析

福建农业学报 ›› 2011, Vol. 26 ›› Issue (6): 947-951.

猪流行性腹泻病毒FJ-11A株的分离与ORF3基因序列分析

陈如敬, 吴学敏, 车勇良, 王隆柏, 魏宏, 庄向生, 严山, 周伦江

福建省农业科学院畜牧兽医研究所/福建省畜禽疫病防治工程技术研究中心,福建福州 350013

收稿日期:2011-09-18 修回日期:2011-11-28 出版日期:2011-12-15 发布日期:2011-12-15
作者简介:周伦江(1973-),男,博士,副研究员,研究方向:动物病毒分子生物学(E-mail:lunjiang@163.com)
基金资助:
国家公益性行业(农业)科研专项(NYHYZX07-034);福建省财政专项——福建省农业科学院科技创新团队建设项目(STIF-Y02)

Isolation of Porcine Epidemic Diarrhea virus strain FJ-11A in Fujian Province and Sequence Analysis of the ORF3 Gene

CHEN Ru-jing, WU Xue-min, CHE Yong-liang, WANG Long-bai, Wei Hong, ZHUANG Xiang-sheng, YAN shan, ZHOU Lun-jiang

Institute of Animal Husbandry and Veterinary Medicine/Fujian Animal Diseases Control Technology Development Center, Fujian Academy of Agricultural Sciences, Fuzhou,Fujian 350013, China

Received:2011-09-18 Revised:2011-11-28 Online:2011-12-15 Published:2011-12-15

摘要/Abstract

摘要： 从福建省某疑似流行性腹泻病毒感染的猪场采集腹泻粪便样品和小肠组织,利用Vero细胞连续传代后,分离到猪流行性腹泻病毒福建株(FJ-11A株)。根据GenBank中登录的猪流行性腹泻病毒ORF3基因特征设计特异性引物,采用RT-PCR方法对猪流行性腹泻病毒福建株ORF3基因进行扩增,将扩增目的片段经胶回收后进行克隆测序。结果表明,所扩增的目的片段基因编码有完整的ORF3开放阅读框,全长为675 bp,编码有224个氨基酸。运用DNAStar软件,将获得的ORF3基因序列和GenBan中登录的猪流行性腹泻病毒株ORF3基因序列进行分析比较和遗传进化分析,猪流行性腹泻病毒福建株ORF3基因和韩国分离株CPF1074和PFF513该基因核苷酸同源性最高,均为99.3%,与疫苗株CV777核苷酸同源性为97.8%。从遗传进化上看,我国PEDV分离株ORF3基因处在同一个分支,而弱毒株则处在相对独立的分支。

关键词: 猪流行性腹泻病毒, ORF3基因, 序列分析

Abstract: Porcine epidemic diarrhea virus (PEDV) FJ-11A strain was isolated from piglets suffering from a severe diarrhea in a pig farm of Fujian Province using Vero cell.The PEDV ORF3 gene was obtained using reverse transcription-polymerase chain reaction (RT-PCR) method according to the sequence download from the GenBank.The results showed that the ORF3 gene of the FJ-11A strain was 675 bp, encoding an open reading frame (ORF) with 224 amino acids.Compared with the ORF3 gene published previously in GenBank by DNAstar software, the ORF3 sequence of FJ-11A strain had the highest nucleotide homology at 99.3% with CPF1074 and PFF513, isolating in South Korea, and also had 97.8% homology with vaccine strain CV777. Based on genetic evolutionary, the PEDV strains which isolated in China were in the same phylogenetic relationship, whereas the candidate vaccine strains (attenuated DR13 and CV777) were in the other phylogenic lineage.

Key words: Porcine epidemic diarrhea virus, ORF3 gene, Genetic analysis

中图分类号:

S 852.65

陈如敬, 吴学敏, 车勇良, 王隆柏, 魏宏, 庄向生, 严山, 周伦江. 猪流行性腹泻病毒FJ-11A株的分离与ORF3基因序列分析[J]. 福建农业学报, 2011, 26(6): 947-951.

CHEN Ru-jing, WU Xue-min, CHE Yong-liang, WANG Long-bai, Wei Hong, ZHUANG Xiang-sheng, YAN shan, ZHOU Lun-jiang. Isolation of Porcine Epidemic Diarrhea virus strain FJ-11A in Fujian Province and Sequence Analysis of the ORF3 Gene[J]. Fujian Journal of Agricultural Sciences, 2011, 26(6): 947-951.

参考文献

[1] 斯特劳 B E,阿莱尔 S D,蒙加林 W L,等.猪病学[M].第8版.北京:中国农业大学出版社,2000:181-187.

[2] PENSAERT M,DEBOUCK P. A new Coronavirus like particle associated with diarrhea in Swine [J]. Arch Virol, 1978, 58: 243-247.

[3] 蔡宝详.介绍几种新近发现的猪传染病[J].畜牧与兽医,1982,(5):218-221.

[4] PURANAVEJA S,POOLPERM P,LERTWATCHARA-SARAKUL P,et al. Chinese-like strain of porcine epidemic diarrhea virus, Thailand[J]. Emerg Infect Dis, 2009, 15(7):1112-1115.

[5] KIM L, HAYES J, LEWIS P, et al. Molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus (TGEV) and porcine respiratory coronavirus (PRCV) field isolates co-circulating in a swine herd [J]. Arch Virol, 2000, 145(6):1133-47.

[6] 邹勇,钱永清,唐永兰,等.猪流行性腹泻、猪传染性胃肠炎和猪轮状病毒RT-PCR鉴别诊断技术研究[J].上海农业学报,2003,19(2):82-84.

[7] 张强敏,郭福生,尹燕博,等.猪流行性腹泻病毒分子生物学特征[J].中国病毒学,2002,17(4):381-384.

[8] 吕茂杰,陈建飞,时红艳,等.猪流行性腹泻病毒核衣壳蛋白与感染细胞核磷蛋白的共定位分析[J].微生物学报,2011,51(5):643-647.

[9] PARK S, MOON H, LUO Y, et al. Cloning and further sequence analysis of the ORF3 gene of wild-and attenuated-type porcine epidemic diarrhea viruses [J]. Virus Genes, 2008, 36 (1):95-104.

[10] CHEN J, WANG C, ShHIH, et al. Molecular epidemiology of porcine epidemic diarrhea virus in China[J]. Arch Virol, 2010,155 (9):1471-1476.

[11] BRIDGEN A, DUARTE M, TOBLER K, et al. Sequence deter mination of the nucleocapsid protein gene of the porcine epidemic diarrhea virus confirms that this virus is a coronavirus related to human coronavirus 229E and porcine transmissible gastroenteritis virus [J]. J Gen Virol, 1993, 74 (9): 1795-1804.

[12] 毛雅元,张桂红,葛俊伟,等.猪流行性腹泻病毒地方株LJB/03分离及培养特性[J].病毒学报,2010,26(6):483-488.

[13] HOFMANN M,WYLER R. Propagation of the virus of PED in cell culture [J]. J Clin Microbial, 1988, 26: 2235-2239.

[14] 张百灵,陈建飞,邢雅玲,等.猪流行性腹泻(PEDV)与抗病毒天然免疫[J].中国生物化学与分子生物学报,2011,27(6):516-523.

[15] 宣华,邢德坤,王殿派,等.应用猪胎肠单层细胞培养猪流行性腹泻的研究[J].中国人民解放军兽医大学学报,1984,4(3):202-208.

[16] KADOI K, SUGIOK H, SATOH T, et al. The propagation of a porcine epidemic diarrhea virus in swine cell lines [J]. New Microbial, 2002, 25:285-290.

[17] KWEON C, LEE J, HAN M, et al. Rapid diagnosis of porcine diarrhea virus infection by polymerase chain reaction [J]. J Vet Med Sci, 1997, 59(3):231-232.

[18] ISHIKAWA K, SEKIGUCHI H, OGINO T, et al. Direct and rapid detection of porcine diarrhea virus by RT-PCR [J]. J Virol Methods, 1997, 69(1-2):191-195.

[19] 钱永清,闻人楚,唐永兰,等.猪流行性腹泻病毒的分离培养鉴定[J].上海农业学报,1999,15(2):41-44.

[20] SEHMIT Z A,TOBERK,SUTER M,et al. Prokaryotic expression of porcine epidemic diarrhea ORF3 [J]. Adv Exp Med Biol, 1998, 440:775-780.