【作者】 张志云；

【导师】 梁爱华；

【作者基本信息】 山西大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 接合生殖是纤毛虫特有的一种有性生殖模式。在接合生殖期间，交配型互补的细胞相互识别配对，进行减数分裂、交换配子核，然后分开。在八肋游仆虫中，这种细胞间的相互作用由识别开始，而识别则由交配型特异性信号物质—信息素所介导。但有关信息素如何介导细胞之间的相互识别及配对，进而促进接合生殖的作用机制仍不清楚。 信息素是一类分子量约为11kDa、内含5对二硫键的酸性蛋白。本研究利用基因工程的方法，体外构建重组表达质粒，探索用不同的启动子和信号肽序列表达八肋游仆虫信息素3蛋白。 首先，对信息素进行胞内融合型表达，构建了系列重组表达质粒pGEX-6P1-G3、pRSetc-G3、pET32M-G3和pMal-c2x-G3。其中工程菌E.coli Bl21／pGEX-6P1-G3在37℃下，经1.0mmol/L IPTG诱导，SDS-PAGE分析，在相应的37kDa处出现明显的表达条带，但融合蛋白主要以包涵体形式存在。 其次，选用分泌型表达载体pExSecl，构建了工程菌E.coli BL21／pExSecl-G3。重组菌在0.4mmol/L IPTG诱导下进行分泌融合表达，融合蛋白的表达量约占菌体总蛋白的23.6％，表达产物ZZ：G3经IgG-sepharose亲和纯化后基本为单一组分。 本实验对游仆虫信息素在大肠杆菌中的表达进行了系统的比较与分析，筛选获得了有效表达信息素的工程菌株，表达产物分别为包涵体与可溶性蛋白。本实验为研究信息素的结构和功能，进而阐明信息素与其受体的识别过程及其诱导交配的作用机制奠定了基础。更多还原

【Abstract】 Conjugation is a sexual model of reproduction, a feature for ciliates. During conjugation, the complementary mating type cells unite pairwise undergo meiosis, exchange gametic nuclei and separate again. For Euplotes octocarinatus, the interaction between cells begins with mutual recognition, which is induced by mating type specific signal substances, the pheromones. However, the molecular mechanism of the process remains unknown.Pheromones are characterized as acidic, cysteinc-rich polypeptides with a molecular mass of about 11 kDa. In the study, several recombinant expression vectors were constructed in vitro by using the technique of gene engineering, making pheromone 3 be expressed under the control of different promoters and signal pcptides.To obtain the fusion pheromone in the E.coli cytoplasm, a series of recombinant vectors are constructed including pGEX-6Pl-G3, pRSetc-G3, pET32M-G3 and pMal-c2x-G3. Among them the engineering strain of E.coli BL21/pGEX-6Pl-G3 was induced with 1.0 mmol/L IPTG at 37 . The results of SDS-PAGE show a prominent band appears at the 37kDa, however, most of the fusion protein exits in the form of the inclusion bodies.To obtain the secret fusion protein in the culture medium, the engineering strain , E.coli BL21/pExSecl-G3, was constructed. The percent of the fusion protein in that of the bacterium bodies is 23.6%, and the produced ZZ:G3 fusion protein present a single band after purified by affinity IgG-sepharose.The systematic comparison and analysis of expression of pheromone 3 of Euplotes in ? coli have been performed in this study. Engineering bacteria, screened for efficient expression of pheromone 3, express the proteins in type of insoluble (inclusion) and soluble forms, respectively. The results of this research provide basal information for studying the structure and function of pheromone, for elucidating the process of recognition between the pheromone and receptor and the mechanism of mating of Euplotes.更多还原