Abstract:

 【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.

Key words: Anopheles sinensis, odorant-binding protein, gene cloning, prokaryotic expression, human odorant, binding affinity