Establishment of a sandwich ELISA system to detect diapause hormone, and developmental profile of hormone levels in egg and subesophageal ganglion of the silkworm, Bombyx mori
Establishment of a sandwich ELISA system to detect diapause hormone, and developmental profile of hormone levels in egg and subesophageal ganglion of the silkworm, Bombyx mori
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DOI：
10.2108/zsj.22.213
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发表时间：
2005-02-01
影响因子：
0.9
通讯作者：
Yamashita, Okitsugu
中科院分区：
文献类型：
--
作者：
Kitagawa, Norio;Shiomi, Kunihiro;Yamashita, Okitsugu
家蚕的滞育激素（DH）是由雌蚕食道下神经节（SG）产生的，通过靶向卵巢诱导胚胎滞育。DH由DH、信息素生物合成激活神经肽（PBAN）和三种其他神经肽（SGNP）组成的前体蛋白加工而成。由于这五种神经肽在C-末端共享一个共同的序列FXPRLamide，因此需要对DH本身进行直接和特异性测定，以了解浓度变化的概况。在这项研究中，我们产生了针对DH的N-末端区域的小鼠单克隆抗体（抗DH [N] mAb），并使用抗DH [N] mAb和针对DH的C-末端的兔多克隆抗体建立了夹心酶联免疫吸附试验。该程序使我们能够在飞摩尔水平（相当于SG的1/10）特异性地定量DH分子。然后，我们绘制了胚胎和胚胎后发育过程中的蛋和SG中的DH水平。DH存在于已经注定用于生产滞育和非滞育卵的晚期胚胎中。在幼虫发育过程中，两种类型的SG中的DH水平逐渐增加，并在蛹早期达到峰值。在蛹中期，滞育产卵型的SG和SG-脑复合体中的DH水平显著降低，从而表明DH主动释放到血淋巴中。从5龄幼虫到成虫，滞育和非滞育产卵型的SG和SG-脑复合体的DH水平没有性别差异。
In the silkworm Bombyx mori, diapause hormone (DH) is produced in the female subesophageal ganglion (SG) and induces embryonic diapause by targeting developing ovaries. DH is processed from a precursor protein consisting of DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides (SGNPs). Because these five neuropeptides share a common sequence, FXPRLamide, at the C-terminus, a direct and specific assay for DH itself is required in order to understand the profile of concentration changes. In this study, we produced a mouse monoclonal antibody (anti-DH[N] mAb) against the N-terminal region of DH and developed a sandwich enzyme-linked immunosorbent assay using the anti-DH[N] mAb and a rabbit polyclonal antibody against the C-terminus of DH. This procedure enabled us to specifically quantify the DH molecule at femtomolar levels (equivalent to 1/10 of SG). We then plotted DH levels in eggs and SGs during embryonic and post-embryonic development. DH was present in late-stage embryos that had been destined for the production of both diapause and nondiapause eggs. DH levels in SG gradually increased in both types during larval development and peaked at the early pupal stage. At the middle pupal stage, DH levels in SG and SG-brain complex decreased markedly in the diapause-egg producing type, thus indicating active release of DH into the hemolymph. From 5th instar larva to adult, no sexual differences in DH levels were observed in SGs or SG-brain complexes from diapause and nondiapause egg-producing types.