核桃举肢蛾性信息素结合蛋白2的分子克隆和免疫荧光定位

›› 2015, Vol. 58 ›› Issue (10): 1054-1062.

核桃举肢蛾性信息素结合蛋白2的分子克隆和免疫荧光定位

李文海, 黄兴龙, 王敦, 冯纪年^*

（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）

出版日期:2015-10-20 发布日期:2015-10-20
作者简介:李文海, 男, 1988年生, 陕西西安人, 硕士研究生, 研究方向为农业昆虫与害虫防治, E-mail: wenhaili@nwsuaf.edu.cn

Molecular cloning and immunofluorescence localization of sex pheromone binding protein 2 from Atrijuglans hetaohei (Lepidoptera: Heliodinidae)

LI Wen-Hai, HUANG Xing-Long, WANG Dun, FENG Ji-Nian^*

(Key Laboratory of Plant Protection Resources & Pest Management of Ministry of Education, Northwest A&F University, Yangling, Shaanxi 712100, China)

Online:2015-10-20 Published:2015-10-20

摘要/Abstract

摘要： 【目的】明确核桃举肢蛾 Atrijuglans hetaohei Yang性信息素结合蛋白2(AhetPBP2)在核桃举肢蛾触角中的分布。【方法】本研究提取羽化后3-4 d的核桃举肢蛾成虫触角总RNA并反转录合成cDNA，设计简并引物进行RT-PCR获得cDNA片段，然后利用RACE技术获得 AhetPBP2全长cDNA序列。将去除信号肽序列的AhetPBP2进行原核表达，重组蛋白经镍柱纯化并免疫新西兰大白兔制备多克隆抗体。制备的多克隆抗体作为一抗对核桃举肢蛾触角进行免疫组化分析。【结果】AhetPBP2基因cDNA序列全长923 bp，开放阅读框504 bp，共编码167个氨基酸，分子量为19.26 kD，等电点为5.47。1 mmol/L IPTG诱导10 h获得可溶性重组蛋白，Western blot结果表明重组蛋白诱导表达成功，ELISA检测显示抗体效价为1:1 024 000。免疫荧光定位结果显示核桃举肢蛾成虫触角部分感器被AhetPBP2抗体标记。【结论】核桃举肢蛾成虫触角的部分感器中可能存在AhetPBP2，推测该部分感器具有感受性信息素的功能。

关键词: 核桃举肢蛾, 性信息素结合蛋白, 原核表达, 多克隆抗体, 免疫荧光定位

Abstract: 【Aim】 The objective of this study is to analyze the distribution of AhetPBP2 in the antenna of Atrijuglans hetaohei. 【Methods】 The cDNA was synthesized with the total RNA extracted from antennae of newly emerged adults of A. hetaohei. The cDNA segment of AhetPBP2 was amplified by RT-PCR with degenerate primers. The full-length cDNA sequence of AhetPBP2 was obtained by RACE. AhetPBP2 without the signal peptide sequence was expressed in Escherichia coli BL21(DE3). Recombinant AhetPBP2 was purified through Ni-chelating affinity chromatography. The polyclonal antiserum was prepared by immunizing a male New Zealand white rabbit with the purified AhetPBP2. Immunohistochemical analysis was performed with the polyclonal antibody of AhetPBP2. 【Results】 The full-length cDNA sequence of AhetPBP2 is 923 bp. Its open reading frame is 504 bp, encoding a protein of 167 amino acids with the molecular weight of 19.26 kD and the isoelectric point of 5.47. After induction with 1 mmol/L IPTG for 10 h, the soluble recombinant protein AhetPBP2 was obtained. Western blot showed that the recombinant protein was expressed successfully. ELISA detection showed that the titer for the polyclonal antibody was 1:1 024 000. Immunohistochemical analysis showed that parts of the antennal sensilla were labeled by AhetPBP2 polyclonal antibody. 【Conclusion】 AhetPBP2 is localized on parts of antennal sensilla of A. hetaohei adults, suggesting that these antennal sensilla may be involved in the perception of sex pheromones.

Key words: Atrijuglans hetaohei, sex pheromone binding protein, prokaryotic expression, polyclonal antibody, immunofluorescence localization

李文海, 黄兴龙, 王敦, 冯纪年. 核桃举肢蛾性信息素结合蛋白2的分子克隆和免疫荧光定位[J]. , 2015, 58(10): 1054-1062.

LI Wen-Hai, HUANG Xing-Long, WANG Dun, FENG Ji-Nian. Molecular cloning and immunofluorescence localization of sex pheromone binding protein 2 from Atrijuglans hetaohei (Lepidoptera: Heliodinidae)[J]. , 2015, 58(10): 1054-1062.

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