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利用粉纹夜蛾(Trichoplusia ni)围食膜蛋白多克隆抗体,从已构建的华北大黑鳃金龟Holotrichia oblita中肠cDNA表达文库中筛选得到1个编码羧酸酯酶的cDNA克隆HoCL1,其开放阅读框(ORF)长1 599 bp,编码532个氨基酸,推导的蛋白质分子质量为59.5 kDa,等电点(pI)为4.5。HoCL1蛋白具有羧酸酯酶的保守结构域:1个二硫键形成的位点和1个丝氨酸活性中心,三联体催化活性中心位于Ser207、Asp333和His422上,不含有氮联糖基化位点和氧联糖基化位点,只含有3个半胱氨酸残基。依据氨基酸序列同源性分析和保守结构域分析,HoCL1属于B类酯酶,与赤拟谷盗Tribolium castaneum羧酸酯酶相似性最高,为35.2%。通过与其他昆虫羧酸酯酶序列比对及构建系统发育树,发现HoCL1羧基端的氨基酸序列保守性低,但靠近N端的活性中心处的氨基酸序列则高度保守,可与赤拟谷盗、异色瓢虫Harmonia axyridis羧酸酯酶聚类在一起。羧酸酯酶HoCL1基因的克隆鉴定为进一步研究该基因在华北大黑鳃金龟体内的表达及功能奠定了基础。
A cDNA clone HoCL1 encoding carboxylesterase was screened from the cDNA library of the midgut of Holotrichia oblita, which was constructed by using Trichoplusia ni polyclonal antibody against peritrophic membrane protein. The reading frame (ORF) is 1 599 bp in length and encodes a protein of 532 amino acids. The deduced protein has a molecular mass of 59.5 kDa and an isoelectric point (pI) of 4.5. The HoCL1 protein has a carboxylesterase conserved domain: one disulfide bond forming site and one serine active site, and the triplet catalytic active site is located on Ser207, Asp333 and His422 and does not contain a nitrogen linked glycosylation site And the oxygenated glycosylation site contains only three cysteine residues. Based on amino acid sequence homology analysis and conserved domain analysis, HoCL1 belongs to class B esterase, which has the highest similarity 35.2% with Tribolium castaneum carboxylesterase. By comparing with other insect carboxylesterase sequences and constructing phylogenetic tree, we found that the amino acid sequence of HoCL1 at the carboxyl terminal is low, but the amino acid sequence near the N-terminal active site is highly conserved. Ladybug Harmonia axyridis carboxylesterase clustered together. The cloning and identification of carboxylesterase HoCL1 gene laid the foundation for further study on the expression and function of this gene.