家蚕脂质运载蛋白基因BmLip32的特征与表达活性分析  

Molecular Characteristics and Expression Analysis of Lipocalin Gene BmLip32 in Bombyx mori

作　　者：牛伟涛[1] 李珊珊[2] 聂作明[1] 陈健[1] 吕正兵[1] 王丹[1] 吴燕[2] 吴怡[2] 高攀[2] 于威[1] 盛清[1] 沈红丹[1] 张耀洲[1] 

机构地区：[1]浙江理工大学生命科学学院生物化学研究所,浙江家蚕生物反应器和生物医药重点实验室,杭州310018 [2]浙江理工大学生命科学学院生物技术系,杭州310018

出　　处：《蚕业科学》2009年第2期264-272,共9页ACTA SERICOLOGICA SINICA

基　　金：浙江省新苗计划项目(编号145300F1A07006);国家重点基础研究发展计划“973”项目(编号2005CB121006);国家高技术研究发展计划“863”项目(编号2007AA02170-3,2007AA100504)

摘　　要：从已构建的家蚕蛹期cDNA文库中筛选得到一条新的cDNA片断,经生物信息学分析发现其编码框具有脂质运载蛋白(lipocalin)保守结构域,命名为BmLip32基因,GenBank登录号为FJ602775。构建融合表达质粒pET-28a-BmLip32后转化大肠杆菌BL21并诱导表达,将纯化的His-BmLip32融合蛋白免疫新西兰大白兔成功制备多克隆抗体,效价大于1∶12 800。RT-PCR检测结果表明BmLip32在家蚕蛹期转录水平最高,在5龄幼虫头部中的转录水平明显高于其它组织。Western blotting分析结果显示BmLip32在5龄幼虫头部大量表达。免疫细胞化学实验显示BmLip32蛋白存在于家蚕Bm5细胞的细胞质中。推测BmLip32基因是家蚕神经系统发育相关的重要调控基因。A cDNA sequence was obtained through screening in a Bombyx mori cDNA library constructed by our laboratory. Blast searches against NCBI database revealed that its coding regions contained a lipocalin conserved domain. We preliminarily nominated this novel gene as BmLip32 （GenBank accession number FJ602775）. The BmLip32 ORF was sub-cloned into the prokaryotic expression vector pET-28a and the fusion protein was successfully expressed in Escherichia coli BL21 by IPTG induction. The purified fusion protein His- BmLip32 was used to immune a New Zealand rabbit and polyclonal antibodies with a titer over 1 ： 12 800 were obtained. RT-PCR amplification indicated that expression of BmLip32 transcript was the highest in pupal stage and was obviously higher in brain than in other tissues of the fifth instar larvae. Western blotting showed that BmLip32 markedly existed in brain compared to other tissues in fifth instar larvae. Immunocytochemistry in Brn5 cells showed that Bin- Lip32 existed only in cytoplasm. It is suggested that BmLip32 could be an important regulatory gene involved in the development of Bombyx mori nervous system.

关 键 词：家蚕 BmLip32基因 表达活性 实时荧光定量PCR 免疫印迹 免疫细胞化学 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]