Prokaryotic expression, polyclonal antibody preparation and expression pattern analysis of venom-allergen proteins from Bursaphelenchus xylophilus
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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-VAP1和Bx-VAP2基因在成虫期表达量较高，Bx-VAP3和Bx-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 kDa ~ 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。 Abstract:Objective Venom-allergen proteins (VAPs) are proteins secreted by pine wood nematode during the process of infesting pine trees. Such proteins may inhibit the defense response of pine trees, thereby facilitating the colonization and spread of pine wood nematodes in pine trees. In this study, the prokaryotic expression, polyclonal antibody preparation and expression pattern analysis of four VAPs were conducted to clarify the structures and functions of the VAPs of B.xylophilus (Bx-VAPs), in order to provide basic support for elucidating the mechanism of this kind of protein in the interaction between pine wood nematode and the host pine. Method Polymerase chain reaction (PCR) was used to amplify the fourBx-VAPs genes, and the expression levels of the fourBx-VAPs genes were detected by Real-time quantitative polymerase chain reaction (RT-qPCR) method. At the same time, the amplified full-length products of the four genes were cloned into the pET32b prokaryotic expression vector separately, and the recombinant plasmid pET-32b-VAPS were constructed. After the identification, the correct recombinant plasmids were transformed into Escherichia coli BL21DE3 for induced expression. The purified Bx-VAPs were used to immunize Balb/c mice respectively, and polyclonal antibodies were obtained after four immunizations; the antibody serum titer was determined by indirect enzyme-linked immunosorbent assay (ELISA); the proteins were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot (WB). Finally, using bioinformatics methods to analyze and predict the physical and chemical properties, secondary structure, surface properties and B cell epitopes of the proteins encoded by these four genes. Result The results showed that there were significant differences in the expression levels of the fourBx-VAPs genes of B.xylophilus during different developmental stages. Among them, the Bx-VAP1 and Bx-VAP2 genes were expressed in high level in the adult stage, and the Bx-VAP3 and Bx-VAP4 genes were expressed in high level in propagative third-larval instar (L3). The constructed recombinant plasmid pET-32b-VAPn induce and express the proteins with a molecular weight between 21 kDa and 31 kDa. The three purified polyclonal antibodies anti-VAP1, anti-VAP2 and anti-VAP3 all have higher effect on the B.xylophilus protein solution specificity, but the anti-VAP4 antibody failed to react with the protein solution ofB.xylophilus. The secondary structures of the four Bx-VAPs are dominated by alph-helix and random coils, all have signal peptides, SCP domains, and no transmembrane domains. Bx-VAP1 has many potential dominant B cell epitopes. Conclusion The protein size induced by recombinant plasmid pET-32b-VAPn is consistent with the predicted protein size. All are expression of inclusion bodies, the prepared polyclonal anti-VAP1、anti-VAP2 and anti-VAP3 have high titer and good specificity. Bx-VAP1 has potential B cell epitope advantages. This study provides experimental materials and basis for further research on the function of B.xylophilus VAPs protein and related pathogenic mechanisms. -
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 2st to 4th instar larvae, A indicates adult. Different lowercase letters indicated 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.xylophilus 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 weight protein Marker.
Figure 3. SDS-PAGE analysis of the expression of recombinant protein in E. coli BL21DE3
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 weight 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基因的亲水/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 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 and tertiary structure 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 usageBx-VAP1-F ATGTTCGGTCTACTGTTAGT 全长克隆
full-length gene cloneBx-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 sitesEX-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 reactionRT-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 
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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 acids分子量
Molecular
weight理论等电点
Theoretical pI信号肽的位置
Signal peptide
location跨膜结构域
Transmembrane
domain磷酸化位点数量
Number of phosphorylation sites丝氨酸
Serine苏氨酸
Threonine络氨酸
ComplexinBx-VAP1 ADG86237.1 204 22 434.86 4.93 17 ~ 18 aa 无 12 17 4 Bx-VAP2 ADG86238.1 206 22 418.83 4.68 17 ~ 18 aa 无 15 15 4 Bx-VAP3 ADG86239.1 202 22 446.27 4.31 18 ~ 19 aa 无 16 11 8 Bx-VAP4 CAD5147609.1 201 22 038.58 5.19 17 ~ 18 aa 无 14 17 7
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