[1]祁光宇,刘斌,赵兴绪.猪流行性腹泻病毒HB2015分离株N基因表达及单克隆抗体的制备[J].江苏农业学报,2018,(01):76-80.[doi:doi:10.3969/j.issn.1000-4440.2018.01.011]
　QI Guang-yu,LIU Bin,ZHAO Xing-xu.Expression of N gene of porcine epidemic diarrhea virus（PEDV） HB2015 strain and preparation of monoclonal antibodies against PEDV[J].,2018,(01):76-80.[doi:doi:10.3969/j.issn.1000-4440.2018.01.011]
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猪流行性腹泻病毒HB2015分离株N基因表达及单克隆抗体的制备()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2018年01期
页码:
76-80
栏目:
畜牧兽医·水产养殖
出版日期:
2018-02-25

文章信息/Info

Title:
Expression of N gene of porcine epidemic diarrhea virus（PEDV） HB2015 strain and preparation of monoclonal antibodies against PEDV
作者:
祁光宇1; 2; 刘斌1; 2; 赵兴绪1
（1.甘肃农业大学动物医学院，甘肃兰州730070；2.中农威特生物科技股份有限公司，甘肃兰州730046）
Author(s):
QI Guang-yu1; 2; LIU Bin1; 2; ZHAO Xing-xu1
(1.College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;2.Agricultural Veterinary Biological Science and Technology Co., Ltd, Lanzhou 730046, China)
关键词:
猪流行性腹泻病毒; N基因; 原核表达; 单克隆抗体; IFA分析
Keywords:
porcine epidemic diarrhea virus; N gene; prokaryotic expression; monoclonal antibody; indirect immunofluorescence assay(IFA) analysis
分类号:
S858.282.65
DOI:
doi:10.3969/j.issn.1000-4440.2018.01.011
文献标志码:
A
摘要:
根据已经发表的猪流行性腹泻病毒N基因序列（GenBank: KX016034.1），设计1对特异性引物(上、下游分别插入Bam HⅠ和 XhoⅠ酶切位点)，通过RT-PCR技术扩增N基因，在测序鉴定正确后经Bam HⅠ和 XhoⅠ双酶切，将其克隆至原核表达载体pET-32a上，获得重组表达质粒pET32a-N。表达的蛋白质经纯化后进行Western blotting鉴定分析。以表达的PEDV-N蛋白作为抗原，免疫BALB/c小鼠，取其脾细胞与SP2/0骨髓瘤细胞进行融合，经间接ELISA筛选及亚克隆，获2株PEDV-N特异性单克隆抗体。对获得的单抗进行Western blotting鉴定和间接免疫荧光试验（IFA）分析，结果显示2株单抗均能与PEDV-N蛋白特异性结合和识别。以上结果表明PEDV-N蛋白在大肠杆菌中成功表达，且成功制备了2株具有生物学活性的特异单抗,为建立PEDV金标试纸诊断方法奠定了基础。
Abstract:
According to the published sequence (GenBank: KX016034.1) of porcine epidemic diarrhea virus（PEDV） N gene, a pair of primers (Bam H I enzyme site was inserted in upstream primers and Xho I enzyme site was inserted in downstream primers) were designed， and N gene was amplified by RT-PCR. The fragment after sequencing identification and vector pET-32a were digested by Bam H I and Xho I, and then the fragment was inserted into prokaryotic expression vector pET-32a to construct recombinant plasmid pET32a-N. The expression protein was purified, and then was identified by Western blotting. Using the N protein of PEDV as the antigen hybridoma cell lines stably secreting monoclonal antibody against N protein of porcine epidemic diarrhea virus were generated by fusing SP2/0 myeloma cells with splenocytes from the immunized BALB/c mice. Two hybridoma cell lines secreting monoclonal antibody suitable for N protein of PEDV were established by indirect ELISA and subcloning. Western blotting identification and IFA analysis of obtained monoclonal antibodies were conducted. The results showed that monoclonal antibodies could specifically bind and identify N protein of PEDV. The above results indicated that PEDV-N protein was successfully expressed in Escherichia coli and specific monoclonal antibodies with biological activity were successfully prepared, which laid the foundation for establishing of colloidal gold strip for PEDV detection.

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备注/Memo

备注/Memo:
收稿日期：2017-06-02 基金项目：甘肃省科技计划项目（1104NKCA167） 作者简介：祁光宇(1978-)，男，辽宁辽阳人，博士，助理研究员,从事分子病毒学与免疫学研究。(E-mail)qiguangyu0931@126.com 通讯作者：赵兴绪，(E-mail)zhaoxx(@gsau.edu.cn
更新日期/Last Update: 2018-03-06