棉铃虫Wnt-1基因克隆、多克隆抗体制备及在滞育和非滞育蛹脑中的表达检测

›› 2015, Vol. 58 ›› Issue (1): 15-21.

棉铃虫Wnt-1基因克隆、多克隆抗体制备及在滞育和非滞育蛹脑中的表达检测

陈伟^1，*，徐卫华²

（1. 广东药学院生命科学与生物制药学院, 广东省生物技术候选药物研究重点实验室, 广州 510006；2. 中山大学生命科学学院, 有害生物控制与资源利用国家重点实验室, 广州 510275）

出版日期:2015-01-20 发布日期:2015-01-20
作者简介:陈伟, 男, 1980年生, 博士, 湖南南县人, 讲师，主要从事昆虫生理生化与分子生物学研究, E-mail:thinker98@163.com

Molecular cloning, preparation of polyclonal antibody and expression detection of Wnt1 in the brain of diapausing and non-diapausing pupae of Helicoverpa armigera (Lepidoptera: Noctuiadae)

CHEN Wei^1,*, XU Wei-Hua²

(1. Guangdong Province Key Laboratory for Biotechnology Drug Candidates, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China; 2. State Key Laboratory of Biocontrol and Institute of Entomology, School of Life Sciences, Sun YatSen University, Guangzhou 510275, China)

Online:2015-01-20 Published:2015-01-20

摘要/Abstract

摘要： 【目的】Wnt1蛋白是Wnt/β-catenin信号通路的重要成员。本研究旨在克隆棉铃虫 Helicoverpa armigera Wnt1基因，制备多克隆抗体，进而从蛋白水平初步研究该蛋白在滞育和非滞育蛹脑中的表达情况。【方法】通过RACE的方法克隆棉铃虫Wnt1基因。根据获取的序列构建Wnt1的真核表达载体并调查其亚细胞定位，同时构建原核表达载体，在大肠杆菌 Escherichia coli BL21(DE3)中进行表达、纯化后免疫兔子，制备多克隆抗体。最后用Western blot的方法检测Wnt1蛋白在两种发育状态蛹脑中的表达情况。【结果】成功克隆了棉铃虫Wnt1基因（GenBank登录号为KJ206240）。Wnt1定位在细胞质，通过镍柱纯化获得了较纯的重组蛋白。制备的Wnt1抗体效价高达1:625 000。Western blot结果表明滞育蛹脑中Wnt1蛋白水平明显低于非滞育蛹脑。【结论】获得了高效价的棉铃虫Wnt1多克隆抗体。我们的结果表明滞育蛹脑中Wnt/β-catenin通路很有可能受到抑制。本研究结果为进一步深入研究棉铃虫Wnt/β-catenin信号通路在棉铃虫发育中的作用奠定了基础。

关键词: 棉铃虫, Wnt1, 滞育, 蛋白纯化, 多克隆抗体

Abstract: 【Aim】 Wnt1 is a key component of Wnt/β-catenin signaling pathway. The objective of this research was to clone the Wnt1 cDNA from Helicoverpa armigera , to prepare the polyclonal antibody against Wnt1 and to investigate the Wnt1 protein levels in the brain of diapausing and non-diapausing pupae by Western blot. 【Methods】 Wnt1 gene was cloned by RACE from H. armigera . Eukaryotic expression vector of Har-Wnt1 was constructed to study its subcellular localization. Prokaryotic expression vector was constructed and the recombinant protein was expressed in Escherichia coli BL21 (DE3). After being purified, the recombinant protein was used to immune New Zealand rabbit. The antibody titer was determined by ELISA. Western blot was used to investigate the Wnt1 protein levels in the brain of H. armigera pupae. 【Results】 Har-Wnt1 cDNA was cloned successfully from H. armigera (GenBank accession number: KJ206240). Har-Wnt1 was found to be exclusively in cytoplasm. After immunizing New Zealand rabbit by recombinant protein, we obtained the antibody against Har-Wnt1 and the titer was estimated as high as 1:625 000 dilution detected by ELISA. The expression levels of Har-Wnt1 were significantly higher in the brain of non-diapausing pupae than in the brain of diapausing pupae. 【Conclusion】 High titer antibody against Har-Wnt1 was obtained. Our results suggest that the Wnt/β-catenin pathway may be activated in non-diapausing pupae and down-regulated in diapausing pupae. This study lays a foundation for further investigating the function of Wnt/β-catenin in the diapause of H. armigera.

Key words: Helicoverpa armigera , Wnt1, diapause, protein purification, polyclonal antibody

陈伟, 徐卫华. 棉铃虫Wnt-1基因克隆、多克隆抗体制备及在滞育和非滞育蛹脑中的表达检测[J]. , 2015, 58(1): 15-21.

CHEN Wei, XU Wei-Hua. Molecular cloning, preparation of polyclonal antibody and expression detection of Wnt1 in the brain of diapausing and non-diapausing pupae of Helicoverpa armigera (Lepidoptera: Noctuiadae)[J]. , 2015, 58(1): 15-21.

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