中华绒螯蟹蜕皮抑制激素(mih)基因的原核表达及其分泌特征的研究-细胞生物学专业论文.docxVIP

摘要摘要中华绒螯蟹(Eriocheir sinensis)是我国重要经济蟹类之一，具有较高的营养价值，然 而在养殖过程中，常出现一龄蟹因蜕皮未遂而成僵蟹现象，给生产带来巨大损失。研究 表明，蜕皮抑制激素在中华绒螯蟹蜕皮过程中起着重要作用。中华绒螯蟹蜕皮抑制激素属于甲壳动物高血糖激素家族(CHH)神经肽，与蜕皮激素 (molt hormone，MH)共同调节着中华绒螯蟹的蜕皮过程。MIH 由中华绒螯蟹 X-器官合 成分泌，其成熟肽包括 75 个氨基酸残基，本实验利用分子生物学技术获得了 MIH 成熟 肽基因序列，并成功构建了表达载体 pET-30a-MIH，将其转入表达菌株 Rosetta 中进行 诱导表达，经纯化后获得的融合表达蛋白用于多克隆抗体的制备，成功制备出了效价高、 特异性强的多克隆抗体。制备出的多克隆抗体用于 MIH 在中华绒螯蟹视上神经节的免疫组织化学研究，免 疫组织化学结果表明眼柄神经分泌细胞中细胞类型 1、2、3、4 都参与了 MIH 的分泌， 并且窦腺也呈现阳性反应。通过体外注射 MIH 抗体，在 2h、4h、6h、8h、12h、24h 后检测中华绒螯蟹体内蜕 皮激素的含量，我们发现在 4h、6h 时其体内蜕皮激素含量明显高于对照组，在 12h 后 实验组与对照组蜕皮激素含量相当，无显著差异，此结果为将来进一步研究其作用机制 奠定基础。关键词中华绒螯蟹 蜕皮抑制激素 多克隆抗体 细胞类型 免疫组织化学IAbsractAbstractChinese mitten crab (Eriocheir sinensis) with high nutritional value is one of the important economic crabs in China. However, molting failure brings great loss during cultural process. Studies showed that molt inhibiting hormone plays an important role during the process of molting in E. sinensis.MIH of E. sinensis, which regulates the process of molting with molt hormone, belongs to the crustacean hyperglycemic hormone neuropeptide family. MIH which has 75 amino acids is produced by X-organ in eyestalk.Using molecular techniques, we got the gene sequence of MIH mature peptide and successfully constructed the expression vector pET-30a-MIH. The pET-30a-MIH was transformed into Rosetta, fusion protein which can be used to prepare polyclonal antibody was obtained after purify. At last we got the polyclonal antibody with high potency and high specificity.The polyclonal antibody was used in the research of immunohistochemical study. We found cell type 1、2、3 and 4 participate in the secretion of MIH and it showed positivereaction in sinus gland.By injection of MIH antibody in vitro, we detected the lever of molt hormone in E. sinensis after 2h、4h、6h、8h、12h、24h respectively, we found that the lever of molt hormoneis significantly higher than the control group at the time of 4h and 6h. After 12h the lever of molt hormone in experimental group is equi