普通烟草铁氧还蛋白Ⅰ的原核表达、抗体制备及在ToMV侵染烟草体内表达分析  

Prokaryotic Expression of Fdn-Ⅰ,Preparation of Rabbit Anti-Fdn-Ⅰ Antibody and Analysis of the Fdn-Ⅰ Levels in ToMV-Infected Tobacco Leaves

作　　者：孙现超[1,2] 李勇[2] 周常勇[1,3] 青玲[1] 

机构地区：[1]西南大学植物保护学院,重庆400716 [2]中国科学院微生物研究所,北京100101 [3]中国农业科学院柑桔研究所国家柑桔工程技术研究中心,重庆400712

出　　处：《中国农业科学》2010年第19期3981-3987,共7页Scientia Agricultura Sinica

基　　金：国家自然科学基金(30900937);中国博士后科学基金(20090450798);中央高校基本科研业务费专项资金(XDJK2009B023)

摘　　要：【目的】构建普通烟草铁氧还蛋白I(ferredoxin I,Fdn-Ⅰ)原核表达载体,表达可溶性Fdn-Ⅰ蛋白,并制备其多克隆抗体,分析Fdn-Ⅰ在ToMV侵染烟草叶片内的表达情况。【方法】以Fdn-Ⅰ全长cDNA为模板,用PCR扩增Fdn-Ⅰ,克隆至原核表达载体pEGX-6p-1,构建Fdn-Ⅰ与GST融合表达载体pEGX-Fdn-Ⅰ,重组质粒转化原核表达菌株BL21,优化GST-Fdn-Ⅰ可溶性表达条件后大量表达蛋白,用GST亲和层析法纯化获得可溶性GST-Fdn-Ⅰ蛋白,免疫家兔制备多克隆抗体。ELISA测定抗体效价,Western印迹法检测抗体的特异性和ToMV侵染前后烟草叶片内Fdn-Ⅰ的表达情况。【结果】在温度为28℃,IPTG诱导浓度为0.3mmol·L-1条件下表达出大小为41.3kD的可溶性融合蛋白。纯化获得约4mg可溶性蛋白,免疫家兔制备了效价为1/6400的多克隆抗体。Western印迹结果表明,该抗体可以与Fdn-Ⅰ的原核和酵母表达产物特异性结合。分析表明Fdn-Ⅰ在ToMV侵染后烟草叶片内含量明显低于其在健康烟草叶片内的含量。【结论】通过Fdn-Ⅰ大肠杆菌中的可溶性表达,制备获得了其高效价特异性多克隆抗体,利用该抗体检测表明ToMV侵染烟草降低了Fdn-Ⅰ的表达。Objective The objective of the study is to construct the prokaryotic expression vector of Fdn-Ⅰ and express the soluble Fdn-Ⅰ in E.coil BL21 for preparation of rabbit anti-Fdn-Ⅰ antibody for the analysis of expression of Fdn-Ⅰ in tobacco leaves infected by ToMV.Method Fdn-Ⅰ was amplified from the full-length cDNA of Fdn-Ⅰ by PCR and cloned into pEGX-6p-1,an expression vector fused with GST,to construct the pEGX-Fdn-Ⅰ.The recombinant plasmid was transformed into E.coil BL21 to express the soluble GST-Fdn-Ⅰ protein in the optimized condition.GST-Fdn-Ⅰ protein was purified with high-affinity GST resin to immunize rabbit for preparing anti-Fdn-Ⅰ antibody.The title was determined by enzyme-linked immunosorbant assay（ELISA）.The specificity of anti-Fdn-Ⅰ antibody and the expression of Fdn-Ⅰ in tobacco leaves infected by ToMV were detected by Western blot method.Result The soluble GST-Fdn-Ⅰ protein with molecular weight 41.3 kD was successfully expressed in E.coil BL21 induced with 0.3 mmol·L-1 IPTG at 28℃.About 4 mg fusion protein was purified and used to immunized rabbit.Anti-Fdn-Ⅰ antibody with the title of 1/6 400 was obtained.Western blotting analysis showed that it could bind with Fdn-Ⅰ specially expressed in E.coil and Yeast.In ToMV infected tobacco leaves,the expression of Fdn-Ⅰ was lower than that in health tobacco leaves.Conclusion The recombinant GST-Fdn-Ⅰ protein in E.coli and its polyclonal antibody with high title and specificity were successfully achieved.The result of Western blot with the polyclonal antibody showed that the expression of Fdn-Ⅰ in tobacco leaves was decreased by the infection of ToMV.

关 键 词：铁氧还蛋白Ⅰ 原核表达 抗体 番茄花叶病毒 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]