猪传染性胃肠炎病毒<em>S</em>基因不同编码区的表达和反应原性鉴定

doi:10.7668/hbnxb.2014.06.012

华北农学报 ›› 2014, Vol. 29 ›› Issue (6): 63-67. doi: 10.7668/hbnxb.2014.06.012

所属专题： 畜牧；生物技术；

猪传染性胃肠炎病毒S基因不同编码区的表达和反应原性鉴定

周宏专^1,2, 覃湘婕^1,3, 杨兵¹, 徐福洲¹, 满坤^1,3, 苏霞¹, 张晓东³, 刘爵¹, 罗绪刚², 王金洛¹

1. 北京市农林科学院畜牧兽医研究所, 畜禽疫病防控技术北京市重点实验室, 北京 100097;
2. 中国农业科学院北京畜牧兽医研究所矿物元素营养研究室, 北京 100193;
3. 北京农业生物技术研究中心, 北京 100097

收稿日期:2014-02-07 出版日期:2014-12-28
通讯作者: 王金洛(1956-),男,四川自贡人,研究员,博士,主要从事畜禽疫病防治研究。
作者简介:周宏专(1982-),男,江苏盐城人,在站博士后,主要从事免疫与动物疫病防治研究。
基金资助:
北京市科技计划项目(No.Z131100003113015);北京市农林科学院博士后基金项目

Prokaryotic Expression and Immunoreactivity of Different Encoding Regions of the Spike Gene of Porcine Transmissible Gastroenteritis Virus

ZHOU Hong-zhuan^1,2, TAN Xiang-jie^1,3, YANG Bing¹, XU Fu-zhou¹, MAN Kun^1,3, SU Xia¹, ZHANG Xiao-dong³, LIU Jue¹, LUO Xu-gang², WANG Jin-luo¹

1. Institute of Animal Science and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, Beijing Key Laboratory for Prevention and Control of Infectious Diseases in Livestock and Poultry, Beijing 100097, China;
2. Mineral Nutrition Research Division, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
3. Beijing Agro-biotechnology Research Center, Beijing 100097, China

Received:2014-02-07 Published:2014-12-28

摘要/Abstract

摘要： 通过原核表达系统表达猪传染性胃肠炎病毒(TGEV)S基因不同编码区,以鉴定不同编码区表达产物与TGEV抗体的结合能力。在对TGEV S蛋白抗原位点分析的基础上,针对S蛋白N端的4个抗原位点C、B、D和A,设计引物分别扩增含C、B位点的TCB片段(411 bp)、含D位点的TD片段(441 bp)和含A位点的TA片段(456 bp),经克隆测序后分别插入原核表达载体pET-32a中进行表达,SDS-PAGE电泳显示各片段均高效表达,表达的TCB片段、TD片段和TA片段融合蛋白大小分别为34.6,35.1,36.1 kDa。Western Blot结果表明,表达蛋白与TGEV感染猪阳性血清和His标签抗体均产生阳性反应。结果显示,3个S基因编码片段均具有与TGEV抗体结合的能力,为进一步鉴定不同编码区的免疫原性及抗原保护能力奠定了基础。

关键词: 猪传染性胃肠炎病毒, S基因, 原核表达, 反应原性

Abstract: To identify immunoreactivity of different encoding regions of the spike (S) gene of Transmissible gastroenteritis virus (TGEV),three encoding regions containing four antigenic sites C,B,D and A located at the N-terminus of S protein was analyzed and expressed by prokaryotic expression system.The fragments TCB (containing antigenic sites C and B,411 bp),TD (containing antigenic site D,441 bp) and TA (containing antigenic site A,456 bp) were amplified by PCR.After cloning and sequence,the fragments were inserted into the prokaryotic expression vector pET-32a respectively.The fusion proteins yielded by TCB,TD and TA recombinant expression plasmids were 34.6,35.1,36.1 kDa respectively.The expressed proteins showed strong positive reaction with TGEV-infected swine serum and His tag antibody by Western Blot.These results indicated that the three encoding regions of S gene had a strong immunoreactivity with TGEV positive antiserum,which will play an essential role in identifying immunogenicity and immunological protection of different encoding regions of the S protein of TGEV.

Key words: Porcine transmissible gastroenteritis virus, Spike gene, Prokaryotic expression, Immunoreactivity

中图分类号:

Q786
S828

周宏专, 覃湘婕, 杨兵, 徐福洲, 满坤, 苏霞, 张晓东, 刘爵, 罗绪刚, 王金洛. 猪传染性胃肠炎病毒S基因不同编码区的表达和反应原性鉴定[J]. 华北农学报, 2014, 29(6): 63-67. doi: 10.7668/hbnxb.2014.06.012.

ZHOU Hong-zhuan, TAN Xiang-jie, YANG Bing, XU Fu-zhou, MAN Kun, SU Xia, ZHANG Xiao-dong, LIU Jue, LUO Xu-gang, WANG Jin-luo. Prokaryotic Expression and Immunoreactivity of Different Encoding Regions of the Spike Gene of Porcine Transmissible Gastroenteritis Virus[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2014, 29(6): 63-67. doi: 10.7668/hbnxb.2014.06.012.

http://www.hbnxb.net/CN/Y2014/V29/I6/63

参考文献

[1] Jones T,Pritchard G,Paton D.Transmissible gastroenteritis of pigs [J].The Veterinary Record,1997,141(16):427-428.
[2] Schwegmann-Wessels C,Zimmer G,Schrder B,et al.Binding of transmissible gastroenteritis coronavirus to brush border membrane sialoglycoproteins [J].Journal of Virology,2003,77(21):11846-11848.
[3] He L,Zhang Y M,Dong L J,et al.In vitro inhibition of transmissible gastroenteritis coronavirus replication in swine testicular cells by short hairpin RNAs targeting the ORF7 gene [J].Journal of Virology,2012,9:176.
[4] Meng F,Zhao Z,Li G,et al.Bacterial expression of antigenic sites A and D in the spike protein of transmissible gastroenteritis virus and evaluation of their inhibitory effects on viral infection [J].Virus Genes,2011,43(3):335-341.
[5] 王龙涛.猪传染性胃肠炎病毒纤突蛋白基因体外克隆表达及鉴定[D].长春:吉林大学,2010.
[6] Gebauer F,Posthumus W P,Correa I,et al.Residues involved in the antigenic sites of transmissible gastroenteritis coronavirus S glycoprotein [J].Virology,1991,183(1):225-238.
[7] Enjuanes L,Sune C,Gebauer F,et al.Antigen selection and presentation to protect against transmissible gastroenteritis coronavirus [J].Veterinary Microbiology,1992,33(1-4):249-262.
[8] 曾作财,许承扬,张晓东,等.猪传染性胃肠炎病毒纤突糖蛋白在转基因玉米中的表达[J].分子植物育种,2012,10(5):535-540.
[9] Sestak K,Zhou Z F,Shoup D I,et al.Evaluation of the baculovirus-expressed S glycoprotein of transmissible gastroenteritis virus (TGEV) as antigen in a competition ELISA to differentiate porcine respiratory coronavirus from TGEV antibodies in pigs [J].Journal of Veterinary Diagnostic Investigation,1999,11(3):205-214.
[10] Torres J M,Alonso C,Ortega A,et al.Tropism of human adenovirus type 5-based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus [J].J Virol,1996,70(6):3770-3780.
[11] Tuboly T,Nagy E.Construction and characterization of recombinant porcine adenovirus serotype 5 expressing the transmissible gastroenteritis virus spike gene [J].The Journal of General Virology,2001,82(Pt 1):183-190.
[12] Smerdou C,Anton I M,Plana J,et al.A continuous epitope from transmissible gastroenteritis virus S protein fused to E.coli heat-labile toxin B subunit expressed by attenuated Salmonella induces serum and secretory immunity [J].Virus Research,1996,41(1):1-9.
[13] Tang L,Li Y.Oral immunization of mice with recombinant Lactococcus lactis expressing porcine transmissible gastroenteritis virus spike glycoprotein [J].Virus Genes,2009,39(2):238-245.
[14] Yin J,Li G,Ren X,et al.Select what you need:a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes [J].Journal of Biotechnology,2007,127(3):335-347.
[15] 殷华平.传染性胃肠炎病毒基因组大片段克隆及其主要抗原片段在伪狂犬病病毒中的表达[D].雅安:四川农业大学,2007.
[16] Reguera J,Ordono D,Santiago C,et al.Antigenic modules in the N-terminal S1 region of the transmissible gastroenteritis virus spike protein [J].The Journal of General Virology,2011,92(5):1117-1126.
[17] Reguera J,Santiago C,Mudgal G,et al.Structural bases of coronavirus attachment to host aminopeptidase N and its inhibition by neutralizing antibodies [J].PloS Pathogens,2012,8(8):e1002859.
[18] Usami Y,Fukai K,Ichikawa Y,et al.Virological and serological studies of porcine respiratory coronavirus infection on a Japanese farm [J].The Journal of Veterinary Medical Science,2008,70(9):929-936.
[19] Costantini V,Lewis P,Alsop J,et al.Respiratory and fecal shedding of porcine respiratory coronavirus (PRCV) in sentinel weaned pigs and sequence of the partial S-gene of the PRCV isolates [J].Archives of Virology,2004,149(5):957-974.

选择文件类型/文献管理软件名称

选择包含的内容

猪传染性胃肠炎病毒S基因不同编码区的表达和反应原性鉴定

Prokaryotic Expression and Immunoreactivity of Different Encoding Regions of the Spike Gene of Porcine Transmissible Gastroenteritis Virus

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	肖士奎, 李芳, 张文婷, 吕淑芳, 史国安, 吴疆, 范丙友. *芍药ACS基因的克隆及其蛋白质原核表达*[J]. 华北农学报, 2022, 37(5): 36-44.
[2]	王亚男, 梁岩岩, 严文培, 张银萍, 李强, 吴秀菊. *北细辛AhOMT1基因的克隆及原核表达分析*[J]. 华北农学报, 2022, 37(4): 62-70.
[3]	连凯琪, 纪爽, 周玲玲, 时志琪, 王飞, 张元臣. 黏虫clip型丝氨酸蛋白酶基因的克隆及原核表达[J]. 华北农学报, 2022, 37(1): 195-201.
[4]	范会芬, 孙天杰, 苏伟华, 肖付明, 张洁, 王冬梅. 大豆GmWRKY50的抗体制备及其蛋白水平表达分析[J]. 华北农学报, 2021, 36(6): 28-34.
[5]	姜楠, 郑倩倩, 李倩文, 涂健, 宋祥军, 邵颖, 刘红梅, 祁克宗. *APEC毒力基因ygeG的原核表达载体构建、表达纯化及鉴定*[J]. 华北农学报, 2021, 36(5): 184-190.
[6]	于天飞, 谢鹏宇, 孙婉姝, 李静, 尹海畅, 黎明, 于志丹. 抗鹅细小病毒NS1蛋白单克隆抗体可变区的原核表达[J]. 华北农学报, 2021, 36(5): 211-215.
[7]	徐欢, 段盛文, 冯湘沅, 郑科, 杨琦, 汪启明, 成莉凤, 彭源德. 苎麻脱胶果胶裂解酶基因的高效表达及序列分析[J]. 华北农学报, 2021, 36(5): 18-23.
[8]	雷其冬, 孙旭东, 徐慧妮. *拟南芥AtTCP4基因克隆及原核表达*[J]. 华北农学报, 2021, 36(5): 24-28.
[9]	李默晓, 卞哲, 周启慧, 刘玉卫, 巩校东, 谷守芹, 韩建民. *玉米大斑病菌StRTG2基因结构、原核表达及表达模式分析*[J]. 华北农学报, 2021, 36(4): 191-196.
[10]	王海波, 李芙蓉, 杨金翠, 高永, 郭俊云. *小桐子磷酸葡萄糖变位酶cPGM与pPGM基因的克隆及原核表达分析*[J]. 华北农学报, 2021, 36(4): 23-30.
[11]	刘庆, 孙廷钊, 盖树鹏, 张玉喜, 刘春英. 牡丹PsZFP1转录因子的特性及表达分析[J]. 华北农学报, 2021, 36(3): 67-73.
[12]	李佳皓, 谢敏秋, 万传银, 左瑞洁, 鲁黎明, 李立芹. *马铃薯转录因子StTCP13基因的原核表达及盐胁迫功能研究*[J]. 华北农学报, 2021, 36(2): 33-39.
[13]	胡月清, 王若仲, 黄志刚, 萧浪涛. *马铃薯转录因子基因StWRKY57的克隆及其蛋白原核表达*[J]. 华北农学报, 2021, 36(1): 10-17.
[14]	刘东, 兰艳平, 张丽, 赵岩, 渠云芳, 黄晋玲. *棉花GhPDP基因原核表达条件的优化*[J]. 华北农学报, 2020, 35(5): 55-61.
[15]	栾庆东, 孙举, 刘均华, 张本清, 尹燕博, 王建琳. 我国主要流行的4种血清型禽腺病毒-Ⅰ群Fiber蛋白的交叉中和研究[J]. 华北农学报, 2020, 35(5): 214-219.

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

猪传染性胃肠炎病毒S基因不同编码区的表达和反应原性鉴定

Prokaryotic Expression and Immunoreactivity of Different Encoding Regions of the Spike Gene of Porcine Transmissible Gastroenteritis Virus

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价