暗黑鳃金龟几丁质脱乙酰酶HpCDA5基因的细胞表达及活性分析

Expression and Characteration of the Chitin Deacetylase 5 Gene from Holotrichia parallela Larvae

cdsRDNA文库免疫筛选到编码暗黑鳃金龟幼虫几丁质脱乙酰酶HpdsRCDA5基因,序列分析表明HpdsRCDA5含有1个几丁质脱乙酰酶结构域,属于GroupdsRV类CDA蛋白。构建重组杆状病毒表达载体pdsRFastdsRBac-HpdsRCDA5,转染昆虫细胞sf9,WesterndsRblot分析表明HpdsRCDA5在昆虫细胞sf9中成功表达42dsRkdsRDa的蛋白。利用qdsRRT-PCR方法分析HpdsRCDA5基因组织表达,结果显示HpdsRCDA5基因在中肠中表达最高,为中肠特异表达蛋白。几丁质结合活性表明HpdsRCDA5蛋白只能被强洗脱剂洗脱,具有很强的几丁质结合活性。本研究通过对暗黑鳃金龟几丁质脱乙酰酶HpdsRCDA5的生化特性研究,为进一步明确HpdsRCDA5的生理功能提供理论依据,并为以HpdsRCDA5蛋白为靶标的暗黑鳃金龟生物防治提供支撑。

The HpCDA5 gene encoding chitin deacetylase was obtained by immune-screening the cDNA library of Holotrichia parallela with an anti-PM proteins polyclonal antiserum. The Hp CDA5 protein has a polysaccharide deacetylase-like domain, belonging to Group VCDA. The HpCDA5 was successfully expressed in insect cells（sf9） as secreted proteins using recombinant baculoviruses（Bac to Bac system）. Western blot analysis showed that the apparent molecular weight for recombinant HpCDA5 were about 42 kDa. q RT-PCR approach revealed that HpCDA5 has high expression in midgut. Subsequent chitin binding assays demonstrated that HpCDA5 had chitin binding affinity. The characterization of recombinant Hp CDA5 laid a foundation for further studies and physiological functions of the Hp CDA5 protein, and provide a theoretical basis for the biocontrol of H. parallela using CDA protein as target.