您的位置:首页  > 论文页面

家蚕Bax inhibitor-1融合Polh在家蚕杆状病毒表达系统中的表达

发表时间:2011-03-15  浏览量:1205  下载量:574
全部作者: 白书婉,舒特俊,陈剑清,张耀洲
作者单位: 浙江理工大学生命科学学院生物化学研究所;浙江省家蚕生物反应器和生物医药重点实验室
摘 要: Bax inhibitor-1 (BI-1)是一种包含多个跨膜结构域的抗凋亡蛋白,是受Bcl2和Bax调控的细胞死亡通路上的一个新型调控因子。从实验室构建的家蚕蛹cDNA文库中检索到一条与小鼠Bax inhibator-1有52%同源性的基因,生物信息学分析后,初步命名为BmBI-1(家蚕Bax inhibator-1),GenBank登录号DN237509.首先利用家蚕杆状病毒多角体蛋白(polyhedrin, Polh)溶解度特性纯化Polh,并免疫小鼠获得多克隆抗体。然后利用Bac-to-Bac系统构建含BmBI-1基因的重组病毒vBmBmBI-1和融合Polh基因的重组病毒vBmPolh-BmBI-1,并感染家蚕BmN细胞,SDS-PAGE和Western blotting分析表明与多角体蛋白融合能促进BmBI-1基因的表达。用重组病毒感染家蚕五龄幼虫及蚕蛹,Western blotting分析表明Polh-BmBI-1基因在五龄幼虫和蚕蛹中成功得到表达。荧光定量PCR结果显示在病毒感染细胞72 h后蛋白表达量最高。
关 键 词: 生物化学与分子生物学;家蚕;Bax inhibitor-1;家蚕杆状病毒表达系统;多角体蛋白
Title: Expression and identification of membrane protein Bax inhibitor-1 in BEVS, tagged with Polh
Author: BAI Shuwan, SHU Tejun, CHEN Jianqing, ZHANG Yaozhou
Organization: Institute of Biochemistry, College of Life Science, Zhejiang Sci-Tech University; Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine
Abstract: The Bax inhibitor-1 (BI-1) is an anti-apoptotic protein that is located in endoplasmic reticulum (ER) membranes. It’s a neotype regulating factor concerned with cell death which controlled by Bcl2 and Bax. When screening silkworm cDNA library, the authors found a gene which has a higher homology with Bax inhibator-1, named BmBI-1 (Bombyx mori Bax inhibator-1) after bioinformatics analysis. Baculoviral polyhedrin was used as a novel fusion partner for facilitating expression of BmBI-1 in Bac-to-Bac baculovirus expression system. The baculovirus transfer vector pFastBac HTb-Polh-BmBI-1 and pFastBac HTb-BmBI-1 were constructed. The recombinant transfer plasmid was transformed into the BmDH10Bac competent cells, and inserted into the bacmid DNA by transposition. The recombinant virus vBmPolh-BmBI-1 and vBmBmBI-1 were constructed.The BmN insect cells were infected with the two kinds of recombinant viruses respectively. The result of Western blotting showed that the BmBI-1 fused with Polh can express in infected BmN cell, but only BmBI-1 can not express. The silkworm larvae and pupa were infected with the recombinant virus vBmPolh-BmBI-1, and the aimed 59 ku protein expressed in pupa and the fifth instars larvae was identified with Western blot. The result of real-time quantitative PCR indicated that the mRNA of Polh-BmBI-1 was highest at 72 h infection by recombinant virus.
Key words: biochemistry and molecular biology; Bombyx mori; Bax inhibitor-1; baculovirus expression vector system; polyhedrin
发表期数: 2011年3月第5期
引用格式: 白书婉,舒特俊,陈剑清,等. 家蚕Bax inhibitor-1融合Polh在家蚕杆状病毒表达系统中的表达[J]. 中国科技论文在线精品论文,2011,4(5):459-468.
 
0 评论数 0
暂无评论
友情链接