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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：1643 下载量：870
| 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. |
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