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采用RT-PCR方法克隆了编码禾谷镰刀菌单端孢酶烯3-O-乙酰转移酶Tri101基因的cDNA序列,并连接到原核表达载体pGEX-4T2上,将获得的重组载体pGEX-4T2/Tri101转化大肠杆菌BL21后用IPTG进行诱导表达。SDS-PAGE和Western blot分析表明,经IPTG诱导后,Tri101基因在大肠杆菌BL21中获得了高效表达,融合蛋白GST-Tri101分子量为75.45 kDa。将该融合蛋白切胶纯化后免疫家兔,制备兔抗GST-Tri101多克隆抗体。经ELISA法测定抗体效价大于1∶256 000。Western blot分析表明制备的抗体与原核细胞体外表达的Tri101蛋白可以特异性结合,表明该抗体的特异性良好。应用该抗体验证了感赤霉病小麦中Tri101基因的表达。兔抗GST-Tri101抗体的成功制备,为进一步研究Tri101的生物学功能、细胞定位以及在其它植物中的表达等奠定了基础。
The cDNA encoding the Tri101 gene of Monoclonal strain 3-O-acetyltransferase of Fusarium graminearum was cloned by RT-PCR and cloned into the prokaryotic expression vector pGEX-4T2. The recombinant vector pGEX-4T2 / Tri101 was transformed into E.coli BL21 and induced with IPTG. SDS-PAGE and Western blot analysis showed that Tri101 gene was highly expressed in E. coli BL21 after IPTG induction. The molecular weight of fusion protein GST-Tri101 was 75.45 kDa. Rabbit anti-GST-Tri101 polyclonal antibody was prepared by immunizing rabbits by gel electrophoresis. Antibody titers were greater than 1: 256,000 by ELISA. Western blot analysis showed that the prepared antibody could specifically bind to the Tri101 protein expressed in vitro by prokaryotic cells, indicating that the antibody has good specificity. This antibody was used to verify the expression of Tri101 gene in the head blight wheat. The successful preparation of rabbit anti-GST-Tri101 antibody laid the foundation for further study of the biological function of Tri101, cell location and expression in other plants.