本文選題：中華絨螯蟹 + 蛻皮抑制激素��； 參考：《河北大學(xué)》2017年碩士論文

【摘要】：中華絨螯蟹(Eriocheir sinensis),俗稱河蟹,屬于節(jié)肢動(dòng)物門,甲殼綱,十足目,是我國(guó)五大經(jīng)濟(jì)蟹類之一。對(duì)甲殼動(dòng)物來(lái)說(shuō),只有通過(guò)不斷的蛻皮才能完成其生長(zhǎng)發(fā)育,而蛻皮是一個(gè)非常復(fù)雜的生理過(guò)程,不僅受到神經(jīng)系統(tǒng)的調(diào)節(jié),而且也受到內(nèi)分泌系統(tǒng)的影響。中華絨螯蟹蛻皮抑制激素(molt inhibiting hormone,MIH)屬于神經(jīng)多肽類激素,在甲殼動(dòng)物蛻皮過(guò)程中發(fā)揮著重要的作用,與蛻皮激素(molt hormone,MH)共同調(diào)節(jié)其蛻皮過(guò)程。研究MIH不但可以更深入了解它對(duì)蛻皮機(jī)制的影響,并且可以對(duì)生產(chǎn)實(shí)踐提出理論性的指導(dǎo)。本實(shí)驗(yàn)利用基因重組技術(shù)成功構(gòu)建了MIH原核表達(dá)載體,并在大腸桿菌BL21(DE3)中進(jìn)行了大量表達(dá)。通過(guò)對(duì)大量表達(dá)的菌體進(jìn)行超聲破碎,并分別純化離心后的上清和包涵體沉淀,發(fā)現(xiàn)原核表達(dá)的MIH重組蛋白以包涵體形式存在;用8M尿素溶解包涵體,并利用鎳柱親和層析法進(jìn)行純化,然后用該蛋白進(jìn)行小鼠免疫,免疫完成后應(yīng)用間接ELISA法檢測(cè)抗血清效價(jià)達(dá)到1:10000以上,說(shuō)明該免疫的小鼠適合用于細(xì)胞融合實(shí)驗(yàn)。細(xì)胞融合實(shí)驗(yàn)中,應(yīng)用間接ELISA方法進(jìn)行陽(yáng)性雜交瘤細(xì)胞檢測(cè),共檢測(cè)到13個(gè)陽(yáng)性孔,其中6個(gè)強(qiáng)陽(yáng)性孔;利用有限稀釋法進(jìn)行亞克隆,通過(guò)3次亞克隆最終篩選到1株穩(wěn)定分泌抗體的單克隆雜交瘤細(xì)胞株,間接ELISA法檢測(cè)效價(jià)達(dá)到了1:51200;用該株細(xì)胞誘生小鼠腹水大量制備單克隆抗體,得到的單克隆抗體效價(jià)達(dá)到了1:102400,濃度為2.8 mg/mL。Western blot實(shí)驗(yàn)結(jié)果證明制備的MIH單抗能夠很好的和中華絨螯蟹內(nèi)源分泌的MIH進(jìn)行結(jié)合。應(yīng)用免疫熒光技術(shù),將制備的單克隆抗體用于MIH在視上神經(jīng)節(jié)中表達(dá)特征的研究,結(jié)果顯示:神經(jīng)分泌細(xì)胞類型1、2、3和4均參與了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.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S917.4

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