| 期刊 |
| 小菜蛾普通气味结合蛋白2基因的原核表达与多克隆抗体制备 徐红星，陈东旭，张雅林，王 敦 |
（西北农林科技大学植保资源与利用教育部重点实验室, 陕西杨凌 712100）
摘要：
本实验提取羽化3 d的小菜蛾Plutella xylostella成虫触角总RNA，反转录合成cDNA，以此为模板PCR扩增出小菜蛾普通气味结合蛋白2基因，大小为492 bp，Blast结果显示与多种昆虫的GOBP2具有较高的同源性。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-GOBP2。IPTG成功诱导pMAL-c4E-GOBP2表达出约60 kDa的融合蛋白。优化诱导条件为3 mmol/L终浓度IPTG、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 : 1.28×105。
关键词： 小菜蛾; GOBP2; 原核表达; 多克隆抗体
Cloning, prokaryotic expression and polyclonal antibody preparation of Plutella xylostella general odorant binding protein 2 (GOBP2) gene
XU Hong-Xing, CHEN Dong-Xu, ZHANG Ya-Lin, WANG Dun*（Ministry Education Key Laboratory of Plant Protection Resources & Pest Management, Northwest A & F University, Yangling 712100, Shaanxi Provine, China)
Abstract: A 492 bp open reading frame of the general odorant binding protein 2 (GOBP2) gene 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 insect. The gene was cloned into pMAL-c4E prokaryotic expressive vectors, and the constructed recombinant plasmids pMAL-c4E-GOBP2 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 pMA-c4E-GOBP2 produced a 60 kDa 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 : 1.28×105. Key words: Plutella xylostella; GOBP2; prokaryotic expression; polyclonal antibody |
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