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本实验提取羽化3d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492bp,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60kDa的融合蛋白。优化诱导条件为3mmol/L终浓度IPTG、6h,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。
In this study, total RNA of the antennae of Plutella xylostella, a member of Plutella xylostella, was extracted from 3d fecundated embryos and cDNA was synthesized by reverse transcriptase. As a template, the common odorant binding protein 2 gene of Plutella xylostella was amplified by PCR, and its size was 492 bp. GOBP2 has high homology. The gene was cloned into the expression vector pMAL-c4E and transformed into the host strain TB1 (DE3) to obtain the monoclonal recombinant plasmid pMAL-c4E-GOBP2. IPTG successfully induced pMAL-c4E-GOBP2 expression of about 60kDa fusion protein. Optimized conditions for the induction of 3mmol / L final concentration of IPTG, 6h, to obtain a large number of soluble protein. The expressed fusion protein was purified by affinity chromatography and immunized New Zealand white rabbits to prepare polyclonal antibodies. ELISA analysis showed that the antibody titer reached 1: 1.28 × 105.