小地老虎几丁质脱乙酰基酶基因的克隆与原核表达

Molecular cloning and prokaryotic expression of chitin deacetylase cDNA sequence from Agrotis ipsilon Hufngel

几丁质脱乙酰基酶(Chitin deacetylase,CDA)可以将几丁质修饰为壳聚糖,在昆虫几丁质代谢中具有重要作用。研究以小地老虎(Agrotis ipsilonHufngel)5龄幼虫虫体为材料提取总RNA,利用RT-PCR和RACE技术,扩增得到小地老虎几丁质脱乙酰基酶基因的cDNA序列。该序列含有2 048个碱基,包括一个1 626个碱基的开放读码框,编码541个氨基酸,分子质量约为61.7 ku,多肽的等电点为5.03。推导得到的氨基酸序列与其他昆虫,尤其是鳞翅目昆虫的几丁质脱乙酰基酶高度同源。所获小地老虎CDA基因的cDNA序列已登录GenBank并获得登录号,登录号为JQ012932。将CDA基因连接到PET21b载体上转入到BL21中诱导表达,SDS-PAGE电泳得到目的蛋白条带。

Chitin deacetylase （CDA） is a kind of chitin modification enzyme in insect which can turn chitin into chitosan. It plays an important role in insect chitin metabolism. Total RNA was isolated from the fifth instar larvae of Agrotis ipsilon Hufngel. CDA cDNA sequence was isolated by RT-PCR and rapid amplification of cDNA ends （RACE）. The cDNA, 2 048 base pairs in length, contained an openreading frame of 1 626 base pairs coding for a polypeptide of 541 amino acid residues with a predicted molecular of 61.7 ku and pl 5.03. The predicted protein shared extensive similarities with those from other insects, expecially the lepidopteran insects. The cDNA sequence has been deposited in GenBank with accession number JQ012932. The CDA coding region was cloned into the expression vector PET21b, and the recombinant CDA protein with six-His tag was successfully expressed in the bacteria BL21.