降解赤霉病菌毒素Tri101基因的原核表达及抗体制备

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。

Prokaryotic expression and antiserum preparation of toxicity reduction gene Tri101 from Fusarium graminearum Schwabe

The Tri101 gene of Fusarium graminearum Schw. was amplified by RT-PCR and ligated to the expression vector, pGEX-4T2. The recombinant plasmid, pGEX-4T2/Tri101 was then transformed into E. coli BL21 strain and induced by IPTG. The results of SDS-PAGE and Western blot analysis showed that the Tri101 gene was highly expressed. The molecular weight of the recombinant fusion protein is 75.45 kDa, consistent with the predicted result. Polyclonal anti-GST-Tri101 rabbit antibody was successfully prepared by using purified Tri101 protein as immunogen. The ELISA titer of antiserum against GST-Tri101 is greater than 1:256 000. Western blot analysis showed that the antiserum could specifically bind to the expressed Tri101 protein. Trichothecene 3-O-acetyltransferase encoded by Tri101 gene in Fusarium-damaged kernels of wheat was successfully detected with the antibody. The rabbit antibody against GST-Tri101 can be further used to study the biological function, localization and expression in other plants of Tri101 gene.