文献类型：期刊文献

中文题名：松材线虫VAPs蛋白的原核表达、多抗制备及表达模式分析

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

作者：冯宇倩[1,2] 王璇[1,2] 李东振[1,2] 张伟[1,2] 理永霞[1,2] 张星耀[1,2]

第一作者：冯宇倩

机构：[1]中国林业科学研究院林业新技术研究所,森林病原整合生物学研究室,北京100091;[2]南方现代林业协同创新中心,南京林业大学林学院,江苏南京210037

年份：2021

卷号：43

期号：9

起止页码：38-50

中文期刊名：北京林业大学学报

外文期刊名：Journal of Beijing Forestry University

收录：CSTPCD;;北大核心:【北大核心2020】；CSCD:【CSCD2021_2022】；

基金：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)。

语种：中文

中文关键词：松材线虫;类毒素过敏原蛋白;原核表达;多克隆抗体;蛋白免疫印迹

外文关键词：Bursaphelenchus xylophilus;venom-allergen proteins;prokaryotic expression;polyclonal antibody;western blot

分类号：S763.7

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21~31 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。
[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 Bursaphelenchus 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 four Bx-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,bioinformatics methods were used 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]There were significant differences in the expression levels of the four Bx-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 induced and expressed 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 had higher effect on the B.xylophilus protein solution specificity,but the anti-VAP4 antibody failed to react with the protein solution of B.xylophilus.The secondary structures of the four Bx-VAPs were dominated by alph-helix and random coils,all had signal peptides,SCP domains,and no transmembrane domains.Bx-VAP1 had 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.