中华蜜蜂蜂毒蛋白酶基因cDNA片段的克隆及序列分析

为从中华蜜蜂蜂毒中扩增蛋白酶(Protease)基因,根据意大利蜜蜂蜂毒蛋白酶基因(GenBank登录号NM_001011584)的保守区设计引物,从中华蜜蜂(Apis cerana cerana)工蜂毒腺中快速抽提总RNA,将从毒腺中扩增出的产物克隆到pGEM-T easy 载体上,导入大肠杆菌JM109,阳性克隆经双酶切鉴定后测序,将测序得到的中华蜜蜂蛋白酶基因与已知的意大利蜜蜂核苷酸序列比较,同源性为95.22%.氨基酸序列分别与意大利蜜蜂、玉米螟、胡蜂、冈比亚按蚊、棉铃象甲虫、埃及斑蚊、热带爪蟾、棕尾别麻蝇、广盐螯虾、亚马逊蝮蛇进行同源性比较,同源性分别为91.71%、46.70%、41.94%、41.21%、39.56%、38.38%、37.35%、36.17%、34.24%、28.49%.本试验首次成功从中华蜜蜂工蜂毒腺中扩增到长度为545 bp、编码蛋白酶的基因片段, 所得序列已提交GenBank,登录号为:EF547156,这为以后克隆该基因全长序列以及利用基因工程技术进行蜂毒产业化开发奠定一定的基础.

Cloning and Sequencing Analysis of Protease Gene from the Venom of Apis cerana cerana

In order to clone the protease gene in the venom from Apis cerana cerana,specific primers to protease gene were designed and synthesized according to Apis mellifera ligustica(NM_001011584).Encoding protease cDNA fragments were amplified by RT-PCR from the total RNA in the venom.The PCR products were ligated into pGEM-T easy vector,which was transformed into E.coli JM109.Positive bacteria clones were screened and identified by PCR method and digested with the double restriction enzyme ECoRI.The se quence of protease gene fragment was also determined and analyzed.Compared with Apis mellifera,the homology of the clone Apis cerana cerana protease was 95.23%.Compared with Apis mellifera ligustica,Ostrinia nubilalis,Polistes dominulus,Anopheles gambiae,Anthonomus grandis,Aedes aegypti,Xenopus tropicalis,Sarcophaga peregrina,Pacifastacus leniusculus,Lachesis stenophrys,the homology of the deduced aminoacid sequence was 91.71%,46.70%,41.94%,41.21%,39.56%,38.38%,37.35%,36.17%,34.24%,28.49%,respectively.The protease gene fragment(EF547156) was successfully cloned whose length was 545 bp from the venom of Apis cerana cerana in the first time.This can lay the groundwork for cloning the full-length gene,and furthermore for the use of honeybee venom by gene engineering technique in the future.