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

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

【目的】明确核桃举肢蛾Atrijuglans hetaohei Yang性信息素结合蛋白2(Ahet PBP2)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c DNA,设计简并引物进行RTPCR获得c DNA片段,然后利用RACE技术获得Ahet PBP2全长c DNA序列。将去除信号肽序列的Ahet PBP2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】Ahet PBP2基因c DNA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 k D,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Ahet PBP2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Ahet PBP2,推测该部分感器具有感受性信息素的功能。

【Aim】The objective of this study is to analyze the distribution of Ahet PBP2 in the antenna of Atrijuglans hetaohei. 【Methods】The c DNA was synthesized with the total RNA extracted from antennae of newly emerged adults of A. hetaohei. The c DNA segment of Ahet PBP2 was amplified by RT-PCR with degenerate primers. The full-length c DNA sequence of Ahet PBP2 was obtained by RACE. Ahet PBP2 without the signal peptide sequence was expressed in Escherichia coli BL21( DE3). Recombinant Ahet PBP2 was purified through Ni-chelating affinity chromatography. The polyclonal antiserum was prepared by immunizing a male New Zealand white rabbit with the purified Ahet PBP2.Immunohistochemical analysis was performed with the polyclonal antibody of Ahet PBP2. 【Results】The full-length c DNA sequence of Ahet PBP2 is 923 bp. Its open reading frame is 504 bp,encoding a protein of 167 amino acids with the molecular weight of 19. 26 k D and the isoelectric point of 5. 47. After induction with 1 mmol / L IPTG for 10 h,the soluble recombinant protein Ahet PBP2 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 Ahet PBP2 polyclonal antibody. 【Conclusion】Ahet PBP2 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.