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基于间接ELISA的鲤血清CyHV-3 pORF65抗体检测方法的建立

马艳平 郝乐 梁志凌 马江耀 李盈 柯浩 刘振兴 曹俊明

马艳平, 郝乐, 梁志凌, 马江耀, 李盈, 柯浩, 刘振兴, 曹俊明. 基于间接ELISA的鲤血清CyHV-3 pORF65抗体检测方法的建立[J]. 南方水产科学, 2018, 14(3): 113-119. doi: 10.3969/j.issn.2095-0780.2018.03.014
引用本文: 马艳平, 郝乐, 梁志凌, 马江耀, 李盈, 柯浩, 刘振兴, 曹俊明. 基于间接ELISA的鲤血清CyHV-3 pORF65抗体检测方法的建立[J]. 南方水产科学, 2018, 14(3): 113-119. doi: 10.3969/j.issn.2095-0780.2018.03.014
Yanping MA, Le HAO, Zhiling LIANG, Jiangyao MA, Ying LI, Hao KE, Zhenxing LIU, Junming CAO. Establishment of CyHV-3 pORF65 antibody detection method by indirect carp serum ELISA[J]. South China Fisheries Science, 2018, 14(3): 113-119. doi: 10.3969/j.issn.2095-0780.2018.03.014
Citation: Yanping MA, Le HAO, Zhiling LIANG, Jiangyao MA, Ying LI, Hao KE, Zhenxing LIU, Junming CAO. Establishment of CyHV-3 pORF65 antibody detection method by indirect carp serum ELISA[J]. South China Fisheries Science, 2018, 14(3): 113-119. doi: 10.3969/j.issn.2095-0780.2018.03.014

基于间接ELISA的鲤血清CyHV-3 pORF65抗体检测方法的建立

doi: 10.3969/j.issn.2095-0780.2018.03.014
基金项目: 国家自然科学基金项目 (31402347);广东省科技计划项目 (2016A020210029);广州市科技计划项目 (201707010216);广东省海洋与渔业厅渔港建设和渔业产业发展专项(A201701C04)
详细信息
    作者简介:

    马艳平(1984 — ),女,博士,助理研究员,从事水产病害学研究。E-mail: mayanping2292@163.com

    通讯作者:

    刘振兴(1981 — ),男,博士,副研究员,从事水产病害学研究。E-mail: liuzhenxing81@126.com

    曹俊明(1962 — ),男,博士,研究员,从事营养免疫研究。E-mail: junmcao@163.com

  • 中图分类号: S 917.1

Establishment of CyHV-3 pORF65 antibody detection method by indirect carp serum ELISA

  • 摘要: 鲤疱疹病毒3型(cyprinid herpesvirus 3,CyHV-3)囊膜蛋白ORF65基因全长1 785 bp,编码594个氨基酸。该研究在稀有密码子分析及信号肽与跨膜结构预测的基础上,将pORF65中的N端信号肽与C端跨膜区段删除后进行密码子优化与基因合成,将合成的截短 ORF65 (truncated ORF65) 插入pET32a (+)载体,构建了pET32a-trunORF65;进一步采用DNAstar、ABCpred、BepiPred 1.0软件预测了pORF65的5个B细胞表位优势区段,以合成的截短ORF65为模板,通过SOE-PCR将5个B细胞表位优势区段编码序列融合后插入pET32a (+)载体,构建了pET32a-modORF65。重组质粒分别转入BL21 (DE3)菌株,经IPTG诱导,SDS-PAGE和Western-blot分析,pET32a-modORF65获得高效表达,表达的融合蛋白分子量为56.4 kD。此外,利用rProtein G亲和层析纯化了锦鲤(Cyprinus carpio haematopterus)血清IgM,免疫小鼠,制备了鼠抗锦鲤IgM多克隆抗体。在上述研究的基础上,将纯化的pORF65作为包被抗原,鼠抗锦鲤IgM多克隆抗体作为检测抗体,建立了间接ELISA方法,该方法可以检测pEGFP-ORF65 DNA疫苗免疫锦鲤后产生的特异性抗体。
  • 图  1  pORF65序列分析结果

    箭头指示信号肽切割位点(SignalP 4.1软件预测),粗体标出跨膜序列(TMHMM软件预测),波浪线标出DNAstar预测的B细胞表位优势区段,阴影显示ABCpred预测的B细胞表位,方框标出BepiPred预测的B细胞表位,斜体标出最终表达的B细胞表位优势区段(5'–3'依次为A、B、C、D、E)

    Figure  1.  Result of sequence analysis of pORF65 protein

    Arrow represents signal peptide cutting site (SignalP 4.1 prediction); bold letters represent transmembrane sequence (TMHMM prediction); wavy lines represent B cell epitope predicted by DNAstar software; shadowed letters represent B cell epitope predicted by BepiPred software; boxes represent B cell epitope predicted by BepiPred software; italic letters represent the five segments encoding the major B cell epitopes predicted by comprehensive analysis of three softwares which were named A, B, C, D and E from 5' to 3'.

    图  2  pET32a-compORF65、pET32a-trunORF65质粒诱导表达结果

    M. 蛋白质Marker;1. pET32a (+)质粒未诱导;2. pET32a (+)质粒诱导;3. pET32a-compORF65质粒未诱导;4. pET32a-compORF65质粒诱导;5. pET32a-trunORF65质粒未诱导;6. pET32a-trunORF65质粒诱导;7. pET32a-modORF65质粒未诱导;8. pET32a-modORF65质粒诱导

    Figure  2.  Result of induced expression of pET32a-compORF65 and pET32a-trunORF65 plasmid

    M. protein Marker; 1. non-induced pET32a (+) plasmid; 2. induced pET32a (+) plasmid; 3. non-induced pET32a-compORF65 plasmid; 4. induced pET32a-compORF65 plasmid; 5. non-induced pET32a-trunORF65 plasmid; 6. induced pET32a-trunORF65 plasmid; 7. non-induced pET32a-modORF65 plasmid; 8. induced pET32a-modORF65 plasmid

    图  3  pORF65抗原表位优势区段融合PCR结果

    M. DNA分子标准;1. pORF65抗原表位优势区段

    Figure  3.  SOE-PCR of pORF65 B cell eptitope segments

    M. DNA Marker; 1. pORF65 B cell eptitope segments

    图  4  pORF65重组蛋白Western-blot分析结果

    M. 蛋白质Marker;1. pET32a-modORF65表达蛋白

    Figure  4.  Western blot analysis of pORF65 recombinant protein

    M. protein Marker; 1. purified protein from pET32a-modORF65

    图  5  锦鲤血清IgM纯化结果

    M. 蛋白Marker;1. 未纯化锦鲤血清;2. 洗涤穿流液;3. 洗脱液

    Figure  5.  Result of purification of koi serum IgM

    M. protein Marker; 1. non purified koi serum; 2. washing fluid; 3. elution fluid

    表  1  ORF65扩增引物序列

    Table  1.   Sequence of ORF65 gene amplification

    引物
    primer
    序列
    sequence
    65Hind3CF CCC AAGCTTGCATGGTCTCGCCGCTCG (下划线为Hind Ⅲ酶切位点)
    65XholCR CCG CTCGAGCTTGATGGTCGCGGCGGCCTTG (下划线为XhoⅠ酶切位点)
    65Hind3TF CCC AAGCTTGCCAAACCGTGGTGTATACC (下划线为Hind Ⅲ酶切位点)
    65XholTR CCG CTCGAGCAGGGTAACTGTGTTGCTG (下划线为XhoⅠ酶切位点)
    65AFHind3 CCC AAGCTTGCTTTATGCCGAGTGATGTG (下划线为Hind Ⅲ酶切位点)
    65AR GCCGTTACGGAACCAAT
    65BF ATTGGTTCCGTAACGGCGGCGGCGGCGGCAGCGCAAGCCTGCGCAGTG
    65BR AACTGTGTGAGCTTCTTG
    65CF CAAGAAGCTCACACAGTTGGCGGCGGCGGCAGCAAGTACTGCCCGAATGAG
    65CR TGCGCTTGTGGCATTGGC
    65DF GCCAATGCCACAAGCGCAGGCGGCGGCGGCAGCTACGGCGTTGGCAACAG
    65DR GGTATTGATGTCCGG
    65EF CCGGACATCAATACCGGCGGCGGCGGCAGCACCACCACCACCACCACAACCACACCG
    65ERXhol CCG CTCGAGGGTAACTGTGTTGCTGGC(下划线为XhoⅠ酶切位点)
     注:65Hind3CF/65XholCR为扩增ORF65全基因引物;65Hind3TF/65XholTR为扩增去掉信号肽与C端跨膜疏水区段的截短ORF65引物;65AFHind3/65AR、65BF/65BR、65CF/65CR、 65DF/65DR、65EF/65ERXhol为融合PCR引物  Note: 65Hind3CF/65XholCR represents ORF65 complete encoding sequence primer; 65Hind3TF/65XholTR represents truncated ORF65 gene primer without signal peptide and transmembrane hydrophobic segment; 65AFHind3/65AR, 65BF/65BR, 65CF/65CR, 65DF/65DR and 65EF/65ERXhol represent SOE-PCR primers.
    下载: 导出CSV
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出版历程
  • 收稿日期:  2017-12-18
  • 修回日期:  2018-01-11
  • 刊出日期:  2018-06-01

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