Abstract:

【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.

Key words: Scleroderma guani, venom, serine protease homologue, prokaryotic expression, expression pattern, phenoloxidase