您的位置：首页 > 论文页面
家蚕多角体蛋白基因在大肠杆菌中的表达
发表时间：2010-04-15 浏览量：1682 下载量：739
| 全部作者： | 黄朱梁，于威 |
| 作者单位： | 宁波大学生命科学与生物工程学院；浙江理工大学生物化学研究所 |
| 摘 要： | 对克隆得到的多角体蛋白基因（polh）进行生物信息学分析，结果表明：该基因序列长为738 bp，编码246个氨基酸，预测分子量为28.85 ku.将polh基因克隆到含6×His标签的原核表达载体pET-28a上，构建重组表达载体pET-28a-polh.重组载体经聚合酶链反应（polymerase chain reaction，PCR）和双酶切鉴定表明重组子构建正确。将该重组子转化到大肠杆菌菌株BL 21中，用终浓度为1 mmol/L 异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，表达产物作SDS-PAGE分析，在35.0 ku(计算后的分子量为32.41 ku)附近的位置有一条特异性蛋白条带，与预期值(融合标签3.56 ku，polh 28.85 ku)相符。进一步采用超声裂解菌体，经12 000 rad/min离心，将分离的上清和沉淀，采用SDS-PAGE分析，结果表明表达的融合蛋白主要存在于沉淀中。 |
| 关 键 词： | 生物化学与分子生物学；家蚕；杆状病毒；多角体蛋白基因 |
| Title： | The expression of Bombyx mori polyhedron gene in Escherichia coli |
| Author： | HUANG Zhuliang, YU Wei |
| Organization： | Faculty of Life Science and Biotechnology, Ningbo University；The Institute of Biochemistry, Zhejiang Sci-Tech University |
| Abstract： | In this paper, the biological information of obtained polyhedron gene was analyzed, the open reading frame containing 738 bp, encoded a polypeptide with 246 amino acids and the predicted molecular weight was 28.85 ku. The fusion protein expression vector pET-28a-polh was constructed and identified by polymerase chain reaction (PCR) and restriction enzyme analysis. The fusion gene was expressed in E.coli cell strain BL 21 and induced by IPTG with final concentration of 1 mmol/L, the expressed product was detected by SDS-PAGE. The results showed that there was a specific band at the site of about 35.0 ku, it just matched with the predicted molecular weight (His-tag was weight of 3.56 ku, and polyhedron was weight of 28.85 ku). Further more, the cells were broke up by ultrasonic and then centrifuged at 12 000 rad/min, the supernatant and sedimentum were collected, respectively. The results of SDS-PAGE analysis revealed that the recombination fusion protein was in the sedimentum. |
| Key words： | biochemistry and molecular biology; Bombyx mori; baculovirus; polyhedron gene |
| 发表期数： | 2010年4月第7期 |
| 引用格式： | 黄朱梁，于威. 家蚕多角体蛋白基因在大肠杆菌中的表达[J]. 中国科技论文在线精品论文，2010，3（7）：677-681. |
1
评论数 0请您登录
暂无评论
下载全文
多维论文