[1]吴玉璐,朱建平,杨 莘,等.猪流行性腹泻病毒N基因的表达及抗原性分析[J].中国预防兽医学报,2013,(04):299-303.[doi:doi: 10.3969/j.issn.1008-0589.2013.04.11]
 WU Yu-lu,ZHU Jian-ping,YANG Shen,et al.Expression and antigenic analysis of porcine epidemic diarrhea virus N gene[J].Chinese journal of preventive veterinary medicine,2013,(04):299-303.[doi:doi: 10.3969/j.issn.1008-0589.2013.04.11]
点击复制

猪流行性腹泻病毒N基因的表达及抗原性分析
分享到:

《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2013年04期
页码:
299-303
栏目:
诊断和检测技术
出版日期:
2013-04-15

文章信息/Info

Title:
Expression and antigenic analysis of porcine epidemic diarrhea virus N gene
文章编号:
1008-0589(2013)04-0299-05
作者:
吴玉璐朱建平杨 莘王亚欣徐彦召虞凌雪于 海刘光清童光志*周艳君*
(中国农业科学院 上海兽医研究所,上海 200241)
Author(s):
WU Yu-lu ZHU Jian-ping YANG Shen WANG Ya-xin XU Yan-zhao YU Ling-xue YU HaiLIU Guang-qing TONG Guang-zhi* ZHOU Yan-jun*
(Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China)
关键词:
猪流行性腹泻病毒 重组N蛋白抗原性分析
Keywords:
porcine epidemic diarrhea virus N protein ELISA
分类号:
S852.65
DOI:
doi: 10.3969/j.issn.1008-0589.2013.04.11
文献标志码:
A
摘要:
为深入了解仔猪腹泻的病因,本研究在不同发病猪场采集了115份临床样品,通过RT-PCR方法对猪流行性腹泻病毒(PEDV)进行检测,结果显示总阳性率为81.7 %。进一步从部分样品中对PEDV的N基因进行克隆和序列分析,结果显示采集的临床样品中各分离株之间的氨基酸同源性高达99 %以上,与代表性病毒株CV777的氨基酸同源性为97.4 %~98.1 %,表明目前流行的PEDV其N基因仍然相对保守的。同时,构建了重组表达载体pGEX-N,经诱导表达显示该重组蛋白呈可溶性表达,重组蛋白大小约为81 ku,western blot 分析结果表明该重组蛋白能够被PEDV阳性猪血清特异性识别。将纯化后的重组N蛋白作为包被抗原,对20份已知背景的临床猪血清抗体进行ELISA检测,结果显示有9份血清为抗体阳性,11份血清呈抗体阴性,与应用RT-PCR方法鉴定抗原的结果符合率为85 %。本研究表达的重组N 蛋白具有良好的抗原性,可以将其作为PEDV血清学诊断的候选抗原。
Abstract:
In recent two years, the outbreaks of porcine epidemic diarrhea epidemic caused heavy economic losses for pig industry in China. To investigate the epidemic of diarrhea, a total of 115 clinical samples were collected from different farms with occurrence of diarrhea and detected by RT-PCR, which showed an 81.7% positive rate for porcine epidemic diarrhea virus (PEDV). In addition, nine of PEDV N genes were amplified and sequenced from positive samples, which exhibited that the homologies of deduced amino acid sequence were up to 99% among the isolates and 97.4% to 98.1% identity with PEDV CV777 reference strain. It indicated that N gene of the current PEDVs were still relatively conservative. Furthermore, the N gene was cloned into pGEX-6p-1 vector for expression in E.coli. The expressed recombinant N protein was about 81 ku and reacted positively with PEDV serum. Moreover, twenty clinical pig sera, identified by RT-PCR, were detected by the ELISA coating with the recombinant PEDV N protein, and 17 samples were positive which were in line with the RT-PCR detection. In conclusion, these results indicated that the recombinant PEDV N protein might be a useful antigen for sera-diagnosis of PEDV infection in pigs.

参考文献/References:

[1]Pensaert M B, de Bouck P. A new coronavirus-like particle associated with diarrhea in swine[J]. Arch Virol, 1978, 58: 243- 247.
[2]Chasey D, Cartwright S F. Virus-like particles associated with porcine epidemic diarrhoea [J]. Res Vet Sci, 1978, 25(2): 255- 256.
[3]Park S J, Kim H K, Song D S, et al. Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field isolates in Korea[J]. Arch Virol, 2011, 156:577-585.
[4]宣华,邢德冲. 应用猪胎肠单层细胞培养猪流行性腹泻病毒的研究[J]. 中国人民解放军兽医大学学报,1984,3:202-208.
[5]Li Zhi-li, Zhu Ling, Ma Jing-yun, et al. Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in south China [J]. Virus Genes, 2012, 45(1): 181-185.
[6]孙东波,冯力,时洪艳,等. 猪流行性腹泻病毒分子生物学研究进展[J]. 动物医学进展,2006,27(10):11-14.
[7]Lee H K, Yeo S G. Cloning and sequence analysis of the nucleocapsid gene of Porcine epidemic diarrhea virus Chinju99 [J]. Virus Genes, 2003, 26(2): 207-212.
[8]Raymond C, Jeannie F S, Brenda G H. Identification of Nucleocapsid Binding Sites within Coronavirus-Defective Genomes [J]. Virology, 2000, 277: 235-249.
[9]Rodak L, Valicek L, Smid B, et al. An ELISA optimized for porcine epidemic diarrhoea virus detection in faeces [J]. Vet Microbiol, 2005, 105(1): 9-17.
[10]Jin S O, Song D S. Comparison of an enzyme-linked immuno- sorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection [J]. J Vet Sci, 2005, 6(4): 349-352.
[11]Rodak L, Valicek L, Nevorankova Z, et al. An ELISA optimized for porcine epidemic diarrhoea virus detection in faeces[J]. Vet Microbiol, 2005, 105(1): 9-17.
[12]Hou Xi-lin, Yu Lin-yu, Liu Jian-zhu. Development and evaluation ofenzyme-linked immunosorbent assay based on recombinant nucleocapsid protein for detection of porcine epidemic diarrhea(PEDV) antibodies[J]. Vet Microbiol, 2007, 123(1-3): 86-92.
[13]Ge Jun-wei, Li Bao-xian, Tang Li-jie, et al. Cloning and seque- nce analysis of the N gene of porcine epidemic diarrhea virus LJB/03 [J]. Virus Genes, 2006, 33(2): 215-219.
[14]孙东波,冯力,时洪艳,等. 猪传染性胃肠炎病毒重组N蛋白抗原间接ELISA抗体检测方法的建立[J]. 中国预防兽医学报,2006,28(5):572-580.

相似文献/References:

[1]葛俊伟,姜艳平,汪 淼,等.猪流行性腹泻病毒S1蛋白在干酪乳杆菌中的分泌表达及免疫原性分析[J].中国预防兽医学报,2009,(04):256.
 GE Jun-wei,JIANG Yan-ping,WANG Miao,et al.Recombinant Lactobacillus casei secretory expression of porcine epidemic diarrhea virus spike protein and the immunogenicity analysis[J].Chinese journal of preventive veterinary medicine,2009,(04):256.
[2]姜艳平,葛俊伟,汪 淼,等.猪流行性腹泻病毒重组核蛋白作为检测抗原的初步应用[J].中国预防兽医学报,2008,(08):587.
 JIANG Yan-ping,GE Jun-wei,WANG Miao,et al.Preliminary application of the recombinant nucleocapsid protein of porcine epidemic diarrhea virus LJB/03 as detecting antigen in diagnostic assays[J].Chinese journal of preventive veterinary medicine,2008,(04):587.
[3]冯 力,孙东波,陈建飞,等.猪流行性腹泻病毒N蛋白单克隆抗体的制备与鉴定[J].中国预防兽医学报,2008,(03):190.
 FENG Li,SUN Dong-bo,CHEN Jian-fei,et al.Preparation and characterization of the monoclonal antibodies against nucleocapsid protein of PEDV[J].Chinese journal of preventive veterinary medicine,2008,(04):190.
[4]陈建飞,冯 力*,时洪艳,等.猪流行性腹泻病毒CH/S株N蛋白基因的遗传变异及其原核表达[J].中国预防兽医学报,2007,(11):856.
 CHEN Jian-fei,FENG Li*,SHI Hong-yan,et al.Genetic variation and prokaryotic expression of N protein gene of porcine epidemic diarrhea virus CH/S strain[J].Chinese journal of preventive veterinary medicine,2007,(04):856.
[5]孙东波,冯 力*,时洪艳,等.猪流行性腹泻病毒S蛋白中和表位区单克隆抗体的制备与鉴定[J].中国预防兽医学报,2007,(11):887.
 SUN Dong-bo,FENG Li*,SHI Hong-yan,et al.Preparation and characterization of the monoclonal antibodies against neutralizing epitope region of PEDV S protein[J].Chinese journal of preventive veterinary medicine,2007,(04):887.
[6]高慎阳,邹运明,李一经*.猪流行性腹泻病毒M基因及其片段原核表达效果分析[J].中国预防兽医学报,2007,(01):27.
 GAO Shen-yang,ZOU Yun-ming,LI Yi-jing*.Prokaryotic expression analysis of recombinant M gene and its truncated M’ fragment of porcine epidemic diarrhea virus[J].Chinese journal of preventive veterinary medicine,2007,(04):27.
[7]张志榜,陈建飞,时洪艳,等.猪流行性腹泻病毒M蛋白单克隆抗体的制备及鉴定[J].中国预防兽医学报,2011,(07):568.
 ZHANG Zhi-bang,CHEN Jian-fei,SHI Hong-yan,et al.Preparation of the monoclonal antibody against membrane protein of porcine epidemic diarrhea virus[J].Chinese journal of preventive veterinary medicine,2011,(04):568.
[8]刘随新,石 达,陈建飞,等.猪流行性腹泻病毒N蛋白核仁定位信号对宿主细胞周期的影响[J].中国预防兽医学报,2013,(03):0.
 LIU Sui-xin,SHI Da,CHEN Jian-fei,et al.Effects on host cell cycle by nucleoli localization signal peptides of porcine epidemic diarrhea virus nucleocapsid protein[J].Chinese journal of preventive veterinary medicine,2013,(04):0.
[9]刘孝珍,陈建飞,时洪艳,等.2011年猪流行性腹泻病毒的遗传变异分析[J].中国预防兽医学报,2012,(03):180.[doi:10.3969/j.issn.1008-0589.2012.03.04]
 LIU Xiao-zhen,CHEN Jian-fei,SHI Hong-yan,et al.Genetic variation analysis of porcine epidemic diarrhea virus isolated in 2011[J].Chinese journal of preventive veterinary medicine,2012,(04):180.[doi:10.3969/j.issn.1008-0589.2012.03.04]
[10]石 达,陈建飞,时洪艳,等.猪流行性腹泻病毒N蛋白与Vero E6核仁蛋白Fibrillarin的亚细胞定位分析[J].中国预防兽医学报,2011,(11):833.[doi:10.3969/j.issn.1008-0589.2011.11.01]
 SHI Da,CHEN Jian-fei,SHI Hong-yan,et al.Subcellular localization analysis between porcine epidemic diarrhea virus nucleocapsid protein and fibrillarin[J].Chinese journal of preventive veterinary medicine,2011,(04):833.[doi:10.3969/j.issn.1008-0589.2011.11.01]

备注/Memo

备注/Memo:
收稿日期:2012-09-29
基金项目:国家生猪现代产业技术体系建设项目(NYCYTX-009)
作者简介:吴玉璐(1987-),女,四川乐山人,硕士研究生,主要从事动物病毒分子生物学研究.
*通信作者:E-mail:gztong@shvri.ac.cn; yjzhouz@shvri.ac.cn
更新日期/Last Update: 2013-04-11