家蚕细胞自噬相关蛋白BmATG8的多克隆抗体制备与组织表达分析  

Preparation of Polyclonal Antibody and Tissue Expression Analysis of BmATG8 in Silkworm(Bombyx mori)

作　　者：杨晓冰[1] 李洪建[1] 吴永鹏[1] 刘庆洪[2] 张敏娟[1] 钱永华[1] 焦锋[1] 

机构地区：[1]西北农林科技大学蚕桑丝绸研究所,陕西杨凌712100 [2]中国农业大学生物学院,北京100193

出　　处：《蚕业科学》2014年第5期818-823,共6页ACTA SERICOLOGICA SINICA

基　　金：现代农业产业技术体系建设专项(No.CARS-35);公益性行业(农业)专项(No.201303057);西北农林科技大学基本科研业务费专项(No.ZD2012008;XNY2013-27)

摘　　要：自噬是细胞固有的一种降解机制,在生物体的生长、发育以及感染免疫、衰老、死亡等过程中发挥重要作用。进行家蚕细胞自噬相关蛋白基因的克隆和重组蛋白表达,分析细胞自噬相关蛋白在5龄幼虫不同组织的表达水平和同一组织中随5龄发育时间的表达变化,有助于从分子水平了解家蚕细胞自噬的功能。根据GenBank登录的家蚕细胞自噬相关蛋白基因Atg8（BmAtg8）的序列设计引物,以家蚕5龄幼虫中肠cDNA为模板克隆了BmAtg8的354 bp编码区序列,并成功将其连入pET32a（＋）质粒中。将重组质粒转化E.coli Rosseta菌株继而诱导表达重组BmATG8蛋白,将该蛋白质纯化后强化免疫新西兰大白兔3次,获得效价达1∶512 000的多克隆抗血清。以此多克隆抗体杂交家蚕组织总蛋白,检测到BmATG8及其与磷脂酰乙醇胺偶联的BmATG8-PE 2条免疫印迹条带。Western杂交表明BmATG8蛋白在家蚕5龄第2天幼虫丝腺、中肠和脂肪体中的表达水平较高,其中在5龄第1～2天和第7～8天丝腺组织的表达水平较高,说明家蚕5龄幼虫这3个组织尤其是这2个时间段的丝腺组织中细胞自噬活性极高,反映出家蚕变态发育前这些组织器官细胞内的代谢活动旺盛。As an inherent degradation mechanism of cell,autophagy plays crucial roles in the processes of growth,development,immune,senescence and death of organisms. Research efforts made to clone silkworm（ Bombyx mori） autophagy associated protein genes,to express the recombinant proteins,and to analyze expressional variations of autophagy associated protein in different larval tissues and different developmental stages would help understand the functions of silkworm cell autophagy at molecular level. In this experiment,primers were designed according to silkworm autophagy related gene Atg8（ BmAtg8） registered in GenBank,and a354 bp coding region of silkworm autophagy related gene BmAtg8 was amplified using cDNA of midgut from the 5th instar silkworm larvae as template and subsequently cloned into pET32a（ ＋） plasmid. The recombinant plasmid was transformed into E. coli Rosseta for induced expression of BmATG8 protein. Rabbit polyclonal antiserum with a titer of1∶ 512 000 was collected after ATG8 protein was purified and injected into New Zealand white rabbit for 3 times.Hybridization of this polyclonal antibody with total proteins of silkworm tissues detected 2 immunoblot bands,i. e.,the band of BmATG8 and the band of phosphatidyl ethanolamine coupled protein BmATG8-PE. Western blot analysis revealed that BmATG8 had relatively high expression levels in silk gland,midgut,and fat body of day 2 larvae of the 5th instar. Especially,it had high expression levels in silk gland of day 1 to day 2 and day 7 to day 8 larvae of the 5th instar.These data reflected high autophagic activity of the 2 developmental stages in the above 3 tissues,which is associated with exuberant cell metabolism before metamorphic stage of silkworm.

关 键 词：家蚕 细胞自噬 BmATG8蛋白 多克隆抗体 组织表达 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q786[农业科学—畜牧兽医]