单端孢酶烯3-O-乙酰转移酶编码基因Tri101的原核表达及多克隆抗体制备

Prokaryotic Expression and Polyclonal Antibody Preparation of Trichothecene 3-O-Acetyltransferase Gene Tri101

【目的】克隆Tri101并通过原核表达获得纯化蛋白,通过免疫SPF大白兔获得高效价、高灵敏度的多克隆抗体,为Tri101蛋白及转Tri101作物的检测提供抗体基础【。方法】以禾谷镰刀菌(Fusarium graminearium)F3210为材料,PCR法获得Tri101并连接至表达载体pET29a(+),将重组表达载体转化大肠杆菌BL21(DE3)后利用IPTG进行诱导表达。利用纯化表达产物制备多克隆抗体。【结果】SDS-PAGE分析结果表明,Tri101在大肠杆菌BL21(DE3)中得到表达,重组蛋白大小为50 kD左右,以包涵体形式存在。利用HisTrap亲和柱纯化重组蛋白,最佳咪唑洗脱浓度为200 mmol.L-1。采用烟草蚀纹病毒蛋白酶切除重组蛋白中的His标签,酶切产物经HisTrap亲和柱再次纯化后免疫SPF大白兔,制备多克隆抗体。经ELISA法检测抗体效价为1﹕12 000,检测灵敏度为0.05×10-6μg.mL-1。Western blotting结果证明制备的多克隆抗体对Tri101具有较好的专一性。【结论】通过Tri101的克隆及原核表达,获得了纯化的融合蛋白,通过免疫动物制备了兔源多克隆抗体,可用于Tri101蛋白及转Tri101小麦的检测。

【Objective】Tri101 was cloned and the purified protein was obtained by prokaryotic expression.Polyclonal antibody of high titer and high sensitivity was obtained by SPF rabbits immunization,thus providing a basis for the establishment of the transgenic plants and products.【Method】Tri101 was obtained from Fusarium graminearium Schwabe F3210 by PCR and connected to the expression vector pET29a（＋）,the recombinant vector was transformed into E.coli BL21（DE3） and induced by IPTG.The results indicated that the recombination expression plasmid was constructed and transformed into E.coli BL21（DE3）.The Tri101 protein was induced by IPTG and used as the antigen to immune the SPF rabbits.【Result】The result of SDS-PAGE showed that the molecular weight of expressed protein was about 50 kD,existing in the form of inclusion body.The expressed protein was purified by HisTrap,and the best imidazole concentration was 200 mmol.L-1.His-tag present in recombinant protein was removed by TEV protease and the digestion product was purified by HisTrap.The purified protein was used to prepare polyclonal antibody.The result of ELISA showed that the titer of rabbit anti-Tri101 antiserum was 1﹕12 000,the detection sensitivity was 0.05×10-6μg.mL-1.The result of Western blotting demonstrated that polyclonal antibody had a very good specificity.【Conclusion】Through Tri101 cloning and prokaryotic expression,the purified fusion protein was prepared by immunizing SPF rabbits.Polyclonal antibody was obtained and used for the establishment for the transgenic wheat and products.