小菜蛾性信息素结合蛋白Ⅰ基因的表达及多克隆抗体制备  

Cloning,Expression and Polyclonal Antibody Preparation of Plutella xylostella Pheromone Binding Protein I

作　　者：陈东旭[1] 于欢[1] 徐红星[1] 张雅林[1] 王敦[1] 

机构地区：[1]西北农林科技大学植保资源与病虫害治理教育部重点实验室,陕西杨凌712100

出　　处：《西北农业学报》2011年第8期182-186,共5页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家自然科学基金(30872033)

摘　　要：提取羽化3 d的小菜蛾成虫触角总RNA,反转录合成cDNA,以此为模板PCR扩增出小菜蛾性信息素结合蛋白基因(PxPBPⅠ),大小为495 bp,Blast结果显示与多种昆虫的PBPⅠ具有较高同源性。将该基因克隆到表达载体pMAL-c4E中,转化宿主菌TB1(DE3),获得单克隆重组质粒pMAL-c4E-PxPBPⅠ。IPTG成功诱导pMAL-c4E-PxPBPⅠ表达出约60 ku的融合蛋白。优化诱导条件为3 mmol/L IPTG、诱导表达6 h,可获得大量可溶性蛋白。表达的融合蛋白通过亲和色谱法纯化、免疫新西兰大白兔制备多克隆抗体。ELISA分析表明制备的抗体效价达1∶512 000。A 495 bp open reading frame of the PxPBPⅠgene was amplified by PCR with cDNA of newly emerged Plutella xylostella antennae as template and sequenced.The result of Blast showed that the cloned gene has high nucleotide sequences homology with other insects.The gene was cloned into pMAL-c4E prokaryotic expressive vectors,and the constructed recombinant plasmids pMAL-c4E-PxPBPⅠ were transformed into the host bacteria E.coli TB1（DE3）.The expressive product induced by IPTG was identified by SDS-PAGE and Western blot analysis.The results showed that the recombinant vector pMAL-c4E-PxPBPⅠproduced a 60ku recombinant protein,which was purified by affinity chromatography and then injected into New Zealand rabbit to induce immune serum（polyclonal antibody）.ELISA analysis showed the titer for the polyclonal antibody was 1：512 000.The prepared antibody provides an important basis for functional analysis on insect PBP I.

关 键 词：小菜蛾 PBPⅠ 原核表达 多克隆抗体 

分 类 号：S433.4[农业科学—农业昆虫与害虫防治]