管氏肿腿蜂毒液丝氨酸蛋白酶同源物SgSPH对寄主血淋巴酚氧化酶活性的影响

Effects of the serine protease homologue SgSPH from the venom of Scleroderma guani(Hymenoptera:Bethylidae)on the phenoloxidase activity in the host hemolymph

【目的】本研究旨在通过克隆表达管氏肿腿蜂Scleroderma guani毒液丝氨酸蛋白酶同源物(serine protease homologue,SPH)基因SgSPH,探索其编码的毒液蛋白对寄主血淋巴酚氧化酶活性的影响。【方法】利用RT-PCR技术克隆管氏肿腿蜂毒液SgSPH基因的开放阅读框(ORF),采用生物信息学软件分析其基因序列结构特征;采用qPCR技术检测该基因在不同发育阶段(卵、低龄幼虫、高龄幼虫、老熟幼虫、吐丝幼虫、黄茧蛹、黑茧蛹和羽化后1-5 d成虫)和雌成虫不同组织(头部、胸部、去除毒液器官的腹部和毒液器官)中的相对表达量;利用载体pSUMO-Mut对该基因进行原核表达,用镍亲和层析柱纯化表达的重组蛋白,采用SDS-PAGE和Western blot对获得的重组蛋白进行鉴定;用酶活性测定方法,分析SgSPH重组蛋白对寄主黄粉虫Tenebrio molitor蛹血淋巴酚氧化酶活性的抑制作用。【结果】克隆获得管氏肿腿蜂毒液SgSPH基因(GenBank登录号:MT920663)的ORF,长798 bp,编码265个氨基酸,其中第1-20位氨基酸为信号肽,理论分子量为30.53 kD,等电点为9.59。多序列比对分析表明,SgSPH与其他寄生蜂毒液丝氨酸蛋白酶和SPH具有较低的氨基酸序列一致性(9%~17%),且缺乏保守的催化三联体。qPCR分析表明,SgSPH基因在管氏肿腿蜂成虫阶段和毒液器官中高表达。SDS-PAGE电泳和Western-blot检测发现,成功表达SgSPH重组蛋白,并纯化得到了高纯度SgSPH重组蛋白。酶活性检测结果显示,SgSPH能抑制寄主黄粉虫蛹血淋巴的酚氧化酶活性。【结论】结果提示管氏肿腿蜂毒液SgSPH具有干扰寄主酚氧化酶级联反应的功能。

【Aim】The aim of this study is to clone and express the venom serine protease homologue(SPH)gene of Sclerotium guani(SgSPH),and to investigate the effect of the venom protein encoded by this gene on the phenoloxidase activity in the host hemolymph.【Methods】The open reading frame(ORF)of venom SgSPH gene was cloned from S.guani by RT-PCR.Its sequence features were analyzed using bioinformatic software.The relative expression levels of the venom SgSPH gene at different developmental stages(egg,early instar larva,late instar larva,mature larva,spinning larva,pupa in yellow cocoon,pupa in black cocoon,and 1-5-day-old adults)and in different female adult tissues(head,thorax,abdomen without venom apparatus and venom apparatus)of S.guani were determined by qPCR.This venom gene was expressed with prokaryotic expression vector pSUMO-Mut.The expressed recombinant protein was purified using Ni-chelating affinity chromatography,and examined by SDS-PAGE and Western blot analysis.The inhibitory effect of the recombinant SgSPH on the phenoloxidase activity in the pupal haemolymph of Tenebrio molitor was measured by enzymatic activity assay.【Results】The ORF of the venom SgSPH gene(GenBank accession number:MT920663)of S.guani was cloned.It is 798 bp in length,encoding 265 amino acids,with the signal peptide consisting of amino acids 1-20,and the predicted protein molecular mass of 30.53 kD and pI of 9.59.Multiple sequence alignment results showed that SgSPH of S.guani shares low amino acid sequence identity(9%-17%)with the serine proteases and SPHs from venoms of other parasitoid wasps,and lacks a conservative catalytic triad.The qPCR results indicated that SgSPH gene was abundantly expressed at the adult stage and in the venom apparatus of S.guani.SDS-PAGE and Western blot analyses showed that the recombinant SgSPH was successfully expressed and the highly purified recombinant SgSPH was obtained.Enzymatic activity assay results showed that the recombinant SgSPH was able to inhibit the phenoloxidase activity in the pupal hemolymph of the host T.molitor.【Conclusion】The results suggest that the venom SgSPH of S.guani can interfere with the host phenoloxidase cascade.