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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。
In order to prepare α-tubulin of Fusarium graminearum, α1- and α2-tubulin genes were amplified by PCR from the cDNA of Fusarium graminearum, cloned into pET30a + expression vector and transformed into host bacteria Rossatta (DE3) pLysS, screening positive clones for protein induced expression. The results of SDS-PAGE and Wes-tern-b1ot showed that the fusion protein mainly existed in the form of inclusion bodies. The molecular weight of the fusion protein was about 52.1 and 55.9 kD. The fusion protein reacted specifically with anti-6 × His monoclonal antibody. Purification of the fusion protein by HisTrapTMHP Columns after washing inclusion bodies and renaturation dialysis, we can get the fusion protein with high purity. This study provides a material basis for in vitro screening of microbubble-targeted fungicides.