[1]刘合义,孙留霞,王进轶,等.牛冠状病毒重组N蛋白间接ELISA检测方法的建立 [J].中国预防兽医学报,2009,(08):618-622.
 LIU He-yi,SUN Liu-xia,WANG Jin-yi,et al.Development of an indirect ELISA for the detectionof Bovine coronavirus using recombinant N protein [J].Chinese journal of preventive veterinary medicine,2009,(08):618-622.
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牛冠状病毒重组N蛋白间接ELISA检测方法的建立


 

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

卷:
期数:
2009年08期
页码:
618-622
栏目:
诊断技术
出版日期:
2009-08-15

文章信息/Info

Title:

Development of an indirect ELISA for the detection
of Bovine coronavirus using recombinant N protein


 

作者:
刘合义孙留霞王进轶刘双翼王 红余丽芸朴范泽侯喜林*
(黑龙江八一农垦大学 动物科技学院,黑龙江 大庆 163319)
Author(s):
LIU He-yi SUN Liu-xia WANG Jin-yi LIU Shuang-yi WANG Hong YU Li-yun PIAO Fan-ze HOU Xi-lin*
(Animal Technology College, Heilongjiang August First Land Reclamation University, Daqing 163319, China)
关键词:
 牛冠状病毒重组N蛋白间接ELISA抗体检测
Keywords:
bovine coronavirus recombinant N protein indirect ELISA antibody detection
文献标志码:
A
摘要:
为建立牛冠状病毒(BCV)检测方法,利用构建的pET30a-N 重组质粒高效表达了BCV重组N蛋白,western blot 检测该重组蛋白具有很好的免疫活性。以纯化的蛋白作为包被抗原,通过方阵试验确定了抗原的最适包被浓度为1.75 μg/mL, 血清最佳稀释倍数为1∶200, 酶标二抗最佳稀释倍数为1∶8 000,建立了检测BCV抗体的间接ELISA方法。用该法对黑龙江一些地区采集到的256份牛血清样品进行检测,结果阳性率为65.23 %,与病毒中和试验方法的检测结果符合率达95.31 %。本研究建立的间接ELISA方法为BCV的检测和区域流行病学调查提供了一种快速简便的血清学诊断方法。
Abstract:
An indirect ELISA was established for the rapid detection of specific bovine coronavirus (BCV) antibodies in cattle using a recombinant N protein as an antigen. The recombinant N protein was expressed from recombinant plasmid pET30a-N in Escherichia coli. Western blot confirmed that the expressed fusion protein could specifically react with antiserum against BCV. This assay was optimized using 1.75 μg/mL N protein as coat antigen, 1∶200 dilution of testing serum and 1∶8 000 dilution of HRP-labeled goat anti-bovine IgG. Test against 256 serum samples from Heilongjiang province by this assay detected 65.23 % positive, which showed 95.31 % agreement with VN test.
 

参考文献/References:

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[5] Sato M ., Akashi H l . Detection of bovine coronavirus by enzymelinked immunosorbent [J] . Microbiol , 1993 , 2:83-87.

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[8] Storz J , Rott R ., Kaluza G . Enhancement of plaque formation and cell fusion of an enteropathogenic coronavirus by trypsin treatment [J] . Infect Immun , 1981 , 31(3):1214-1222.

[9] Takahashi E,Inaba Y, Sato K, et al. Epizootic diarrhea of adult cattle associated with a coronavirus-like agent[J]. Vet Microbiol 1980;5:151–154.

[10] 姚火春,杜念兴, 徐为燕 , .牛冠状病毒的血清流行病学调查[J].南京农业大学学报,1990l3(2)l17-121.

[11] Harry V.,Gert-J,J.Rossen. Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes[J].The EMBO Journal,1996,15(8):2020-2028.

[12] Savithra D S, Martin A H, Joanne L.,et al. The Nucleocapsid Protein Gene of Bovine Coronavirus Is Bicistronic[J].Journal of virology Sept.,1992:66(9):5277-5283.

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备注/Memo

备注/Memo:
收稿日期:2008-10-29
基金项目:黑龙江农垦总局攻关课题(HNKXIV-08-07)
作者简介:刘合义(1981-),男,河南淮阳人,硕士研究生,从事动物病毒学研究.
*通信作者:Email:xly_hou@yahoo.com.cn
更新日期/Last Update: 2009-10-26