中华绒螯蟹蜕皮抑制激素单抗的制备及其在眼柄视上神经节中的表达特征

发布时间：2018-06-11 22:38

本文选题：中华绒螯蟹 + 蜕皮抑制激素　；参考：《河北大学》2017年硕士论文

【摘要】：中华绒螯蟹(Eriocheir sinensis),俗称河蟹,属于节肢动物门,甲壳纲,十足目,是我国五大经济蟹类之一。对甲壳动物来说,只有通过不断的蜕皮才能完成其生长发育,而蜕皮是一个非常复杂的生理过程,不仅受到神经系统的调节,而且也受到内分泌系统的影响。中华绒螯蟹蜕皮抑制激素(molt inhibiting hormone,MIH)属于神经多肽类激素,在甲壳动物蜕皮过程中发挥着重要的作用,与蜕皮激素(molt hormone,MH)共同调节其蜕皮过程。研究MIH不但可以更深入了解它对蜕皮机制的影响,并且可以对生产实践提出理论性的指导。本实验利用基因重组技术成功构建了MIH原核表达载体,并在大肠杆菌BL21(DE3)中进行了大量表达。通过对大量表达的菌体进行超声破碎,并分别纯化离心后的上清和包涵体沉淀,发现原核表达的MIH重组蛋白以包涵体形式存在;用8M尿素溶解包涵体,并利用镍柱亲和层析法进行纯化,然后用该蛋白进行小鼠免疫,免疫完成后应用间接ELISA法检测抗血清效价达到1:10000以上,说明该免疫的小鼠适合用于细胞融合实验。细胞融合实验中,应用间接ELISA方法进行阳性杂交瘤细胞检测,共检测到13个阳性孔,其中6个强阳性孔;利用有限稀释法进行亚克隆,通过3次亚克隆最终筛选到1株稳定分泌抗体的单克隆杂交瘤细胞株,间接ELISA法检测效价达到了1:51200;用该株细胞诱生小鼠腹水大量制备单克隆抗体,得到的单克隆抗体效价达到了1:102400,浓度为2.8 mg/mL。Western blot实验结果证明制备的MIH单抗能够很好的和中华绒螯蟹内源分泌的MIH进行结合。应用免疫荧光技术,将制备的单克隆抗体用于MIH在视上神经节中表达特征的研究,结果显示:神经分泌细胞类型1、2、3和4均参与了MIH的分泌。
[Abstract]:Eriocheir sinensis, belonging to Arthropoda, crustacea, Decapoda, is one of the five major economic crabs in China. For crustaceans, the growth and development of crustaceans can only be accomplished through continuous molting, and molting is a very complex physiological process, which is not only regulated by the nervous system, but also affected by the endocrine system. Mitten sinensis (Eriocheir sinensis), a molt inhibiting hormone, is a neuropeptide hormone, which plays an important role in the molting process of crustaceans. The study of MIH can not only deeply understand the influence of MIH on molting mechanism, but also provide theoretical guidance for production practice. In this experiment, the prokaryotic expression vector of MIH was successfully constructed by gene recombination technique and expressed in large quantities in E. coli BL21 (DE3). By ultrasonic fragmentation of a large number of expressed bacteria and purification of the supernatants and inclusion bodies after centrifugation, it was found that the MIH recombinant protein expressed in prokaryotic cells existed in the form of inclusion bodies, and the inclusion bodies were dissolved with 8m urea. The immunized mice were purified by nickel column affinity chromatography and then immunized with the protein. The titer of antiserum detected by indirect Elisa was over 1: 10000, which indicated that the immunized mice were suitable for cell fusion test. In cell fusion assay, indirect Elisa was used to detect the positive hybridoma cells, 13 positive holes were detected, 6 of them were strongly positive, and the subclones were subcloned by finite dilution method. A monoclonal hybridoma cell line with stable secreting antibody was screened by three subclones, and the titer of indirect Elisa was 1: 51200.Monoclonal antibody was produced from ascites of mice induced by the cell line. The monoclonal antibody titer reached 1: 102400, and the concentration was 2.8 mg / ml Western blot. The results showed that the prepared monoclonal antibody could bind well with the endogenous MIH secreted by Eriocheir sinensis (Eriocheir sinensis). Using immunofluorescence technique, the prepared monoclonal antibodies were used to study the expression of MIH in the supraoptic ganglion. The results showed that the neurosecretory cell types 1 and 4 were involved in the secretion of MIH.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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