摘要
为了制备小麦赤霉病菌的α-微管蛋白,以小麦赤霉病菌cDNA为模板,PCR扩增出α1-、α2-微管蛋白基因,将其克隆到pET30a+表达载体上,转化到表达宿主菌Rossatta(DE3)pLysS,筛选阳性克隆,进行蛋白诱导表达。SDS-PAGE及Wes-tern-b1ot结果表明:融合蛋白主要以包涵体形式存在;融合蛋白分子量约为52.1和55.9 kD;融合蛋白能与抗6×His的单抗发生特异性反应。通过对包涵体洗涤及透析复性后采用HisTrapTMHP Columns对融合蛋白进行纯化,可以得到纯度较高的融合蛋白。该研究为体外筛选以微管蛋白为靶标的杀菌剂提供了物质基础。
The paper aimed to express the （tubulins of Fusarium graminearum in Escherichia coli and purify them. The （tubulin genes were amplified from F. graminearum cDNA and cloned to the vector pET30a , then transformed into the host Rossatta （DE3）pLysS. After the positive clones were screened by the colony PCR and double digestion, the induced fusion proteins were obtained and verified by SDS-PAGE and Western blot. The positive clones which could express more fusion protein were screened, however, the fusion proteins formed were mainly inclusion bodies. The molecular weight of fusion proteins were confirmed to be 52.1 and 55.9 kD by SDS-PAGE, which also showed specific activity to anti-6 x His monoclonal antibody. After washing inclusion bodies with the buffer containing 2 and 3 mol/L urea, the purity of fusion protein would increase. The soluble fusion protein was obtained by dialysis to binding buffer and then a-tubulins were purified by HisTrapTM HP Columns. The purified tubulins can be used in the studies of new fungicide screening that target tubulin in vitro.
出处
《植物病理学报》
CAS
CSCD
北大核心
2012年第3期252-259,共8页
Acta Phytopathologica Sinica
基金
国家自然科学基金(30971891)
国家863计划项目(2008AA10Z414)
