【Aim】 This study aims to carry out prokaryotic expression, antibody preparation and expression profiling of the odorant binding protein OBP6 in Athetis dissimilis, so as to facilitate the future study on the function of AdisOBP6. 【Methods】Bioinformatics software was used to analyze the structure characteristics of AdisOBP6 protein. AdisOBP6 antibody was prepared by immunizing New Zealand white rabbits four times using the recombinant protein obtained by prokaryotic expression. Fluorescence quantitative PCR and Western blot were used to detect the expression profiles of AdisOBP6 in the antennae of female and male adults, testes at different developmental stages and unfertilized and fertilized eggs of A. dissimilis. 【Results】 Homologous modeling prediction showed that AdisOBP6 has six conserved cysteine residues and seven α-helical structures, which are folded into a binding pocket. The prokaryotic expression results showed that the recombinant protein was expressed most highly and stably under induction with 0.5 mmol/L IPTG at 20℃. Antibody titers exceeded 1∶512 000, 1∶512 000, 1∶512 000, and 1∶64 000, respectively, in four immune replicates. The specific binding of the obtained antibody to AdisOBP6 protein was detected by Western blot. Fluorescence quantitative PCR results showed that the expression level of AdisOBP6 in the testis was much higher than that in the antennae. AdisOBP6 began to express in the testis of A. dissimilis at the larval stage, with a higher expression level in the larval testis than in the pupal testis, and its expression level gradually reached the peak in the testis of adults after eclosion, but it was scarcely expressed or expressed at a very low level in unfertilized eggs and fertilized eggs. Western blot analysis showed that AdisOBP6 protein was also mainly expressed in the testis, and its expression level was very low in the antennae of female and male adults, fertilized eggs and unfertilized eggs. 【Conclusion】 In this study, the prokaryotic expression of AdisOBP6 was realized, and its antibody was successfully prepared. This study also confirmed that AdisOBP6 is highly expressed in the testis, and the expression level of AdisOBP6 reaches the peak after adult eclosion, suggesting that AdisOBP6 may be involved in insemination process. These results lay a foundation for further studying the function of AdisOBP6.

 【Aim】 This study aims to clone the odorant binding protein 2 gene AsinOBP2 of Anopheles sinensis, and to analyze the expression profiles of AsinOBP2 and the binding affinities of its recombinant protein with human odorants. 【Methods】 The full-length cDNA sequence of AsinOBP2 was cloned by RT-PCR and RACE techniques. The expression levels of AsinOBP2 in different developmental stages (female and male pupa, and 0-, 1-, 2, 3-, 6- and 9-day-old adults), different chemosensory tissues (antenna, proboscis and maxillary palp) of the 3-day-old adult, and the 3-day-old female adults before and post blood meal were measured by qPCR. The recombinant protein AsinOBP2 was expressed and purified by prokaryotic expression and affinity chromatography, respectively. The binding affinities of the recombinant AsinOBP2 with 39 human odorants were analyzed using fluorescence competitive binding assay. 【Results】 The full-length cDNA of AsinOBP2 (GenBank accession no.: MT700441) was successfully cloned and sequenced. Its ORF is 492 bp in length, encoding 163 amino acids with the signal peptide of 30 amino acids at the N-terminus. The mature protein possesses six conserved cysteines and belongs to the Classic OBP subfamily. The qPCR results showed that the expression level of AsinOBP2 in adults gradually increased with the eclosion time, peaked on the 6th day after eclosion, and then went down. AsinOBP2 was mainly expressed in the antenna of female adult mosquito. The expression level of AsinOBP2 in the 3-day-old female adult decreased significantly after blood meal. The results of fluorescence competitive binding assay showed that among the 39 human odorants tested, the recombinant AsinOBP2 had binding activities with 12 compounds. AsinOBP2 had a higher binding affinity to indole, with the dissociation constant Ki value of 27.15 μmol/L, followed by n-propylamine, 2methylbutanal and decanal with the Ki values of 42.49, 48.33 and 47.90 μmol/L, respectively. 【Conclusion】 Based on the expression profiles of AsinOBP2 and the binding affinities of its recombinant protein to human odorants, which indicated that AsinOBP2 has obviously selective binding properties to human odorants, we inferred that AsinOBP2 plays an important role in host-seeking behavior of female mosquito.

【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.