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松材线虫VAPs蛋白的原核表达、多抗制备及表达模式分析

冯宇倩 王璇 李东振 张伟 理永霞 张星耀

冯宇倩, 王璇, 李东振, 张伟, 理永霞, 张星耀. 松材线虫VAPs蛋白的原核表达、多抗制备及表达模式分析[J]. 北京林业大学学报, 2021, 43(9): 38-50. doi: 10.12171/j.1000-1522.20210202
引用本文: 冯宇倩, 王璇, 李东振, 张伟, 理永霞, 张星耀. 松材线虫VAPs蛋白的原核表达、多抗制备及表达模式分析[J]. 北京林业大学学报, 2021, 43(9): 38-50. doi: 10.12171/j.1000-1522.20210202
Feng Yuqian, Wang Xuan, Li Dongzhen, Zhang Wei, Li Yongxia, Zhang Xingyao. Prokaryotic expression, polyclonal antibody preparation and expression pattern analysis of venom-allergen proteins from Bursaphelenchus xylophilus[J]. Journal of Beijing Forestry University, 2021, 43(9): 38-50. doi: 10.12171/j.1000-1522.20210202
Citation: Feng Yuqian, Wang Xuan, Li Dongzhen, Zhang Wei, Li Yongxia, Zhang Xingyao. Prokaryotic expression, polyclonal antibody preparation and expression pattern analysis of venom-allergen proteins from Bursaphelenchus xylophilus[J]. Journal of Beijing Forestry University, 2021, 43(9): 38-50. doi: 10.12171/j.1000-1522.20210202

松材线虫VAPs蛋白的原核表达、多抗制备及表达模式分析

doi: 10.12171/j.1000-1522.20210202
基金项目: 中央级公益性科研院所基本科研业务费专项(CAFYBB2020SZ008),国家自然科学基金青年项目(31901317)
详细信息
    作者简介:

    冯宇倩,博士,助理研究员。主要研究方向:松材线虫致病机理。Email:fengyuqian1988@163.com 地址:100091北京市海淀区香山路东小府1号中国林科院分析楼

    责任作者:

    理永霞,副研究员。主要研究方向:森林病理学。Email:liyongxiaxjs@163.com 地址:同上

  • 中图分类号: SS763.7

Prokaryotic expression, polyclonal antibody preparation and expression pattern analysis of venom-allergen proteins from Bursaphelenchus xylophilus

  • 摘要:   目的   类毒素过敏原蛋白(VAPs)是松材线虫侵染松树过程中分泌的一类蛋白。此类蛋白可通过抑制松树的防卫反应,从而利于松材线虫在松树内的定殖与扩散。本研究对4种类毒素过敏原蛋白进行原核表达、多抗制备和基因的表达模式分析,明确松材线虫Bx-VAPs蛋白的结构与功能,为阐明该类蛋白在松材线虫与寄主松树互作中的作用机理提供基础支撑。   方法   本研究利用聚合酶链式反应(PCR)扩增松材线虫4个Bx-VAPs基因,通过实时荧光定量(RT-qPCR)方法检测不同龄期松材线虫4个Bx-VAPs基因的表达量。同时,将扩增的4个基因全长产物分别转化至pET32b原核表达载体,构建重组质粒pET-32b-VAPs,经鉴定正确后的重组质粒转化至大肠杆菌BL21DE3进行诱导表达。利用纯化的Bx-VAPs蛋白分别免疫Balb/c鼠,4次免疫后获得多克隆抗体;采用间接酶联免疫吸附法ELISA测定抗体血清效价;利用聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹法(Western-blot)进行蛋白鉴定。最后,应用生物信息学方法分析这四个蛋白的理化性质、二级结构和表面特性,预测B细胞抗原表位。   结果   松材线虫4个Bx-VAPs基因在不同龄期的表达量存在显著差异,其中Bx-VAP1Bx-VAP2基因在成虫期表达量较高,Bx-VAP3Bx-VAP4基因在繁殖型L3时期表达量较高。构建获得的重组质粒pET-32b-VAPn诱导表达的蛋白分子量介于21 ~ 31 kDa之间,纯化后的多克隆抗体anti-VAP1、anti-VAP2和anti-VAP3均对松材线虫蛋白液具有较高的特异性,但anti-VAP4抗体未能与松材线虫蛋白液结合反应。4个Bx-VAP蛋白的二级结构均以α螺旋和无规则卷曲为主,存在信号肽,具有SCP结构域,无跨膜结构域,Bx-VAP1潜在的优势B细胞抗原表位较多。   结论   重组质粒pET-32b-VAPn诱导表达的蛋白大小与预测的蛋白大小一致,均为包涵体表达,制备的多克隆抗体anti-VAP1、anti-VAP2和anti-VAP3效价高,特异性良好,Bx-VAP1具有潜在的B细胞抗原表位优势,为进一步研究松材线虫VAPs蛋白的功能及其相关致病机制研究提供了实验材料和基础。

     

  • 图  1  Bx-VAPs基因PCR扩增的电泳图

    Figure  1.  Electrophoresis map of PCR amplification of Bx-VAPs gene

    图  2  不同发育阶段松材线虫Bx-VAPs的相对表达量

    E代表卵,L2 ~ L4 代表繁殖型2 ~ 4龄幼虫,A代表成虫。不同小写字母表示不同发育阶段基因表达量差异显著性(P < 0.05)。下同。E indicates egg stage, L2 − L4 indicate propagative 2nd to 4th instar larvae, A indicates adult. Different lowercase letters indicate significant differences in gene expression at different developmental stages (P < 0.05). The same below.

    Figure  2.  Relative expression level of Bx-VAPs in B. ylophilus at various developmental stages

    图  3  重组蛋白在大肠杆菌BL21DE3中表达的SDS-PAGE分析

    1. 无IPTG诱导的BL21DE3-BxVAPn全菌;2. IPTG诱导的BL21DE3-BxVAPn 全菌;3. 上清液;4. 包涵体;5. 纯化的BxVAPn蛋白;6. 低分子量蛋白标记。1, BL21DE3–BxVAP1 without IPTG; 2, BL21DE3- BxVAP1 induced with IPTG; 3, supernatant; 4, inclusion body; 5, purified BXVAP1 protein; 6, low molecular mass protein marker.

    Figure  3.  SDS-PAGE analysis of the expression of recombinant protein in E. coli BL21DE3

    图  4  间接ELISA测定抗体效价

    Figure  4.  Determination of antibody titer by indirect ELISA

    图  5  Western blot验证抗体的特异性

    M. 低分子量蛋白质标记物;Bx. 松材线虫蛋白液。M, low molecular mass protein marker; Bx, protein solution of B. xylophilus.

    Figure  5.  Western blot verifying antibody specificity

    图  6  Bx-VAPs蛋白生物信息学分析

    A. Bx-VAPs的氨基酸序列;B. Bx-VAPs蛋白的亲水/疏水性;C. Bx-VAPs蛋白系统发育树。A, amino acid sequence of Bx-VAPs; B, predicted hydrophilicity/ hydrophobicity of Bx-VAPs; C, phylogenetic tree analysis of Bx-VAPs protein.

    Figure  6.  Bioinformatics information analysis of the Bx-VAPs protein

    图  7  松材线虫 Bx-VAPs蛋白结构域的预测

    Figure  7.  Predicted domains in Bx-VAPs of B. xylophilus

    图  8  Bx-VAPs的三级结构(A)和二级结构(B)预测图

    h代表α-螺旋,c代表无规则卷曲,e代表延伸链,t代表β转角。h represents α-helix, c represents random coil, e represents extended strand, and t represents β turn.

    Figure  8.  Secondary structure (B) and tertiary structure (A) prediction diagram of Bx-VAPs

    图  9  Bx-VAPs蛋白的B细胞抗原表位预测

    A. Bx-VAP1蛋白的B细胞表位预测;B. Bx-VAP2蛋白的B细胞表位预测;C. Bx-VAP3蛋白的B细胞表位预测;D. Bx-VAP4蛋白的细胞表位预测。A, B cell epitope prediction of Bx-VAP1 protein; B, B cell epitope prediction of Bx-VAP2 protein; C, B cell epitope prediction of Bx-VAP3 protein; D, B cell epitope prediction of Bx-VAP4 protein.

    Figure  9.  B cell antigenic epitope prediction of Bx-VAPs protein

    表  1  引物信息

    Table  1.   Primer information

    引物名称
    Primer name
    引物序列
    Primer sequence
    引物用途
    Primer usage
    Bx-VAP1-F ATGTTCGGTCTACTGTTAGT 全长克隆
    Full-length gene clone
    Bx-VAP1-R TCAGGCGCTACACAGACTGT
    Bx-VAP2-F ATGGTTCGAGTATTAGTTCT
    Bx-VAP2-R TCAGGCACTGCACAAACTGT
    Bx-VAP3-F ATGTTCCGAGTTCTCCTGAC
    Bx-VAP3-R CTATTCGGCACTGCAGAGTG
    Bx-VAP4-F ATGATAACTAAATTCCTGTG
    Bx-VAP4-R TTAAGCGACGCACAATCCCT
    EX-VAP1-F GTATTTTCAGGGATCCGAATTCGACAGGTTCAGCAACAGCCAA 添加双酶切位点
    Addition of double cleavage sites
    EX-VAP1-R GGTGGTGGTGGTGGTGCTCGAGTCAGGCGCTACACAGACTGT
    EX-VAP2-F GTATTTTCAGGGATCCGAATTCACCAAATTCAGCGAAAGCCAA
    EX-VAP2-R GGTGGTGGTGGTGGTGCTCGAGTCAGGCGCTACACAGACTGT
    EX-VAP3-F GTATTTTCAGGGATCCGAATTCGCCAGGTTAAGCGACGATGAG
    EX-VAP3-R GGTGGTGGTGGTGGTGCTCGAGCTATTCGGCACTGCAGAGTG
    EX-VAP4-F GTATTTTCAGGGATCCGAATTCGAAAAGTTGAATAGCGCTGAG
    EX-VAP4-R GGTGGTGGTGGTGGTGCTCGAGTTAAGCGACGCACAATCCCT
    RT-VAP1-F AATGTGATGGAGGCTGAC 实时荧光定量PCR
    Real-time quantitative polymerase chain reaction
    RT-VAP1-R GCTACACAGACTGTCACT
    RT-VAP2-F GCTGTGCCATTCAGAACT
    RT-VAP2-R GCTCCCATCATGTTTCCA
    RT-VAP3-F TTGGACCTTCACCGTCTG
    RT-VAP3-R CTTCTTCTTCCTCGCACTG
    RT-VAP4-F CTCCGACCTGGAAGACAA
    RT-VAP4-R CGCTCTCATGGCAATGTT
    β-actin -F TCCGTACCCTGAAGTTGGCTAACC
    β-actin -R AAGTGGAGACGAGGGAATGGAACC
    下载: 导出CSV

    表  2  松材线虫的Bx-VAPs蛋白序列及理化性质

    Table  2.   Protein sequence and physicochemical properties of Bx-VAPs in B. xylophilus

    蛋白名称
    Protein name
    基因库登录号
    GenBank ID
    氨基酸数
    Number of
    amino acid
    分子量
    Molecular
    mass/Da
    理论等电点
    Theoretical pI
    信号肽的位置
    Signal peptide
    location/aa
    跨膜结构域
    Transmembrane
    domain
    磷酸化位点数量
    Number of phosphorylation site
    丝氨酸
    Serine
    苏氨酸
    Threonine
    络氨酸
    Complexin
    Bx-VAP1 ADG86237.1 204 22 434.86 4.93 17 ~ 18 无 No 12 17 4
    Bx-VAP2 ADG86238.1 206 22 418.83 4.68 17 ~ 18 无 No 15 15 4
    Bx-VAP3 ADG86239.1 202 22 446.27 4.31 18 ~ 19 无 No 16 11 8
    Bx-VAP4 CAD5147609.1 201 22 038.58 5.19 17 ~ 18 无 No 14 17 7
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-05-31
  • 修回日期:  2021-07-15
  • 网络出版日期:  2021-08-20
  • 刊出日期:  2021-10-15

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