松材线虫VAPs蛋白的原核表达、多抗制备及表达模式分析

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...

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

  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-VAP_S 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-VAP_n 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-VAP_n 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.