Cloning and expression of a fat body-specific chitinase cDNA from the spider, Araneus ventricosus
Cloning and expression of a fat body-specific chitinase cDNA from the spider, Araneus ventricosus
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DOI：
10.1016/j.cbpc.2004.11.009
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发表时间：
2005-03-01
影响因子：
2.2
通讯作者：
Jin, BR
中科院分区：
文献类型：
--
作者：
Han, JH;Lee, KS;Jin, BR
从大腹蜘蛛中克隆了脂肪体特异的几丁质酶基因。华支睾吸虫几丁质酶(AvChit1)的编码区全长1515bp，开放阅读框(ORF)长431个氨基酸。AvChit1具有几丁质酶家族18个活性位点的特征和一个N-糖基化位点。推导的AvChit1基因的氨基酸序列与粘虫几丁质酶和人几丁糖苷酶的同源性为43%，与一些缺乏丝氨酸/苏氨酸和几丁质结合区的昆虫几丁质酶的同源性为30-40%。基因组DNA的Southern杂交分析表明，AvChit1基因以单拷贝形式存在。Northern和Western印迹分析及酶活性分析表明，AvChit1在腹主动脉脂肪体中具有组织特异性表达。重组AvChit1在杆状病毒感染的昆虫Sf9细胞中以61 kDa的多肽形式表达，以0.1%乙二醇几丁质为底物进行几丁质酶活性测定。用N-糖基化的特异性抑制剂衣霉素处理重组病毒感染的Sf9细胞，发现AvChit1是N-糖基化的，但糖基不是几丁质降解活性所必需的。(C)2004 Elsevier Inc.保留所有权利。
A fat body-specific chitinase cDNA was cloned from the spider, Araneus ventricosus. The cDNA encoding A. ventricosus chitinase (AvChit1) is 1515 bp long with an open reading frame (ORF) of 431 amino acid residues. AvChit1 possesses the chitinase family 18 active site signature and one N-glycosylation site. The deduced amino acid sequence of AvChit1 cDNA showed 43% identity to both Glossina morsitans morsitans chitinase and a human chitotriosidase, and 30-40% to some insect chitinases which lack both the serine/threonine and chitin binding domains. Southern blot analysis of genomic DNA suggested the presence of AvChit1 gene as a single copy. Northern and Western blot analysis and enzyme activity assay showed the tissue-specific expression of AvChit1 in the A. ventricosus fat body. The AvChit1 cDNA was expressed as a 61 kDa polypeptide in baculovirus-infected insect Sf9 cells and the recombinant AvChit1 showed activity in the chitinase enzyme assay using 0.1% glycol chitin as a substrate. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that AvChit1 is N-glycosylated, but the carbohydrate moieties are not essential for chitinolytic activity. (C) 2004 Elsevier Inc. All rights reserved.