禾谷镰孢菌β-微管蛋白基因的克隆及在原核细胞中的表达

Cloning and prokaryotic expression of the β-tubulin genes of Fusarium graminearum

分别扩增了禾谷镰孢菌(Fusarium graminearum)对多菌灵(MBC)敏感菌株(MBCS)的β1-微管蛋白基因(β1-S)和β2-微管蛋白基因(β2-S),及多菌灵中抗菌株(MBCMR)和高抗菌株(MBCHR)的β2-微管蛋白基因(β2-MR和β2-HR),测序正确后连接到pET32a(+)原核表达载体上,转化至大肠杆菌Rosepta感受态中,经IPTG(1 mmol.L-1)诱导,得到了N末端融合6×His纯化标签的表达产物。SDS-PAGE电泳分析表明:在大肠杆菌中表达了相对分子质量约68×103的蛋白,和预测蛋白大小一致,表达的产物主要以包涵体的形式存在。表达的蛋白经HisTrapTM HP Columns纯化并做了Western-blot验证,结果表明,该蛋白能与抗His标签的单抗发生特异性反应。

β-tubulin gene（β1-S）was amplified from the carbendazim（MBC）-sensitive isolate（MBCS）,so was the β2-tubulin gene from the MBCS（named β2-S）,the β2-tubulin gene from MBC-middle resistant isolate（MBCMR）（named β2-MR）,and β2-tubulin gene from MBC-high resistant isolate（MBCHR）（named β2-HR）,and after sequenced,the target genes were connected to the prokaryotic expression vector pET32a（＋）,and then transformed to the strain Rosepta.The recombinant strains were induced by IPTG（1 mmol·L-1）,and the proteins with 6×His-tagged were extracted,and SDS-PAGE electrophoresis analysis showed that the molecular weight of the proteins expressed in E.coli was about 68×103,as expected.And they were mainly present in inclusion bodies.After purified with HisTrapTM HP Columns and Western-blot,the expressed proteins had a specific reaction with the monoclonal antibody anti-His tag.