中华蜜蜂蜂毒镇静肽基因的cDNA克隆和表达  

Cloning and Expression of a cDNA Encoding Secapin from the Venom of Apis cerana cerana

作　　者：张素方[1] 施婉君[1] 张传溪[1] 程家安[1] 

机构地区：[1]浙江大学应用昆虫学研究所,杭州310029

出　　处：《中国生物化学与分子生物学报》2003年第3期343-348,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金部分资助项目 (No .3 0 2 710 0 8)~~

摘　　要：从中华蜜蜂 (Apisceranacerana)工蜂毒腺中快速抽提总RNA ,用RT PCR扩增得到大小约为2 5 0bp的cDNA片段 ,测序得到的片段长度为 2 34bp ,为蜂毒前镇静肽原 (preprosecapin)基因编码区的cDNA .以 3′RACE方法 ,扩增和测定了 3′端非编码区 2 19bp序列 .中蜂前镇静肽原cDNA序列与已报道的欧洲意蜂该基因cDNA序列具有 92 %同源性 ,氨基酸序列具有 87%同源性 .代表成熟肽镇静肽的最后 2 5个氨基酸序列 ,中蜂与意蜂同源性为 88% .3′端非编码区cDNA序列与欧洲意蜂序列有 73 1%同源性 .将中华蜜蜂蜂毒镇静肽成熟肽编码区与 3′非编码区部分克隆 ,构建了镇静肽与谷胱甘肽转移酶融合表达的载体pGEX AcSecapin .将载体转化大肠杆菌BL2 1(DE3)进行融合表达 .表达产物与抗GST抗体在 2 9kD处有很强的交叉反应 .大肠杆菌超声破碎后的上清液用SDS PAGE检测到表达的蛋白多为可溶性融合蛋白 。A cDNA fragment encoding preprosecapin was amplified by RT PCR from the total RNA of venom gland of workers of the Chinese honeybee, Apis cerana cerana The PCR product was ligated into pGEM  T easy vector and the nucleotide sequence was analyzed. The fragment was 234 bp in length, containing an ORF coding the preprosecapin. The preprosecapin of A.cerana cerana shared 92% and 87% homology with the European honeybee, A.mellifera preprosecapin in nucleotide and amino acid sequence, respectively. The secapin of the Chinese honeybee shared 88% homology with the European honeybee in amino acid sequence. The 3′ noncoding region of the gene was also amplified by 3′RACE from the total RNA of Chinese honeybee. The sequence of the 3′ noncoding region shared 73 1% homology with the European honeybee. The coding region of the matured peptide (secapin) together with the 3′ noncoding region was sub cloned into the prokaryotic expression vector pGEX 4T 2. The vector was then introduced into E.coli BL21 (DE3) for expression. A GST fusion protein of 29 kD was detected with anti GST antibody by Western blotting. The SDS PAGE showed that most of recombinant protein was soluble. The secapin was obtained by cleaving the fusion protein with thrombin protease.

关 键 词：中华蜜蜂 蜂毒 镇静肽基因 CDNA克隆 表达 序列分析 

分 类 号：Q963[生物学—昆虫学] Q78