【作者】 王大磊；

【导师】 侯连生；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 盘基网柄菌(Dictyostelium discoideum)是一种低等的单细胞真核生物,在营养丰富的条件下能够依靠吞噬细菌为食,其繁殖生长是通过二分裂的方式。当食物逐渐耗尽后,单个细胞逐渐聚集成多细胞体,历经细胞聚集阶段、细胞丘阶段和蛞蝓体阶段、最终发育成一个由孢子细胞和柄细胞组成的多细胞有机体—子实体阶段。在合适条件下,孢子细胞能够萌发,从而开始新的生命周期。盘基网柄菌的基因组是单倍体,不会由于等位基因的存在而掩盖单基因的突变,因此隐性性状很容易鉴别,拥有非常丰富的突变型细胞。盘基网柄菌发育周期短,整个周期约需24h,各阶段的特征也很清楚,且涉及到一系列生化过程与高等生物十分相似。由于具有以上优点,盘基网柄菌可以作为非常好的模式动物,在研究细胞与细胞间的相互作用以及细胞黏附分子对发育的影响中发挥重要作用。盘基网柄菌中lagC基因编码的gp150蛋白是一种细胞表面粘附分子,对多细胞发育和细胞分化极为重要。gp150缺失的细胞株AK127的发育只能停留在细胞松散聚集阶段,不能完成多细胞发育。由此推测,gp150蛋白在盘基网柄菌发育过程中起着重要作用。在细胞分化过程中,gp150蛋白的分布存在着差异：在分化成孢子细胞的前孢子区内基本没有gp150蛋白分布；而在分化成柄细胞的前柄细胞区则有gp150蛋白分布,并随着发育进程逐渐增加。粘附分子gp150能传递细胞内外的信号,但是目前为止只有少数关于gp150蛋白参与盘基网柄菌细胞内相关信号通路的报道,其具体的信号通路至今仍未完全清楚。为此我们克隆表达了gp150蛋白C末端,并用纯化的目的蛋白制备多克隆抗体,通过免疫共沉淀技术得到多个与gp150分子相关的蛋白,其中两个经质谱鉴定分别为40S ribosomal protein S3 (RPS3)蛋白和40S ribosomal protein S24蛋白(RPS24)。本论文将含有gp150蛋白c末端基因的pET-32a载体转化入表达菌株大肠杆菌BL21(DE3)中,利用lmmol/L的IPTG诱导表达,最终得到表达量较高的存在于包涵体中的融合蛋白。用8mol/L的尿素溶解包涵体后,裂解液经NI-NTA亲合层析纯化融合蛋白,最终得到的gp150融合蛋白纯度达90%以上。利用纯化得到的gp150蛋白作为抗原免疫家兔,从而获得含有抗gp150蛋白的多克隆抗体的抗血清。用Western blot检测抗体的特异性,抗血清能特异地和目的蛋白结合,而对照血清与抗原没有杂交带；用间接ELISA法测定抗体的效价,达到1：128000以上,这证明我们制备的多克隆抗体具有较高的效价和较好的特异性,为下一步的研究工作提供了工具。为了探索盘基网柄菌中与gp150蛋白相互作用的其他蛋白,我们以发育至16h的盘基网柄菌为材料,在非变性的条件下提取总蛋白,将制备的抗gp150的多克隆抗体及Protein A-Agarose加入到总蛋白提取物中。4℃振荡过夜,离心分离抗原抗体复合物,经SDS-PAGE电泳分析得到七条相关蛋白条带,其分子量大约在30KD-15KD左右。我们从中选取了较为清晰的两条条带(分子量分别为25KD和14KD左右)进行质谱分析鉴定,结果分别为40S ribosomal protein S3 (RPS3)蛋白和40S ribosomal protein S24蛋白(RPS24)。通过查阅相关文献,我们发现两蛋白在细胞周期调控、细胞发育及凋亡过程中有着重要作用。实验结果提示在盘基网柄菌多细胞发育中gp150蛋白可能与RPS3和RPS24发生相互作用,借此调节了盘基网柄菌细胞的发育与分化过程。本论文的研究为进一步研究盘基网柄菌粘附分子gp150在胞内信号转导中的作用奠定了基础,也为研究盘基网柄菌整个信号通路做出了贡献。更多还原

【Abstract】 Dictyostelium discoideum is a unicellular eukaryote, which can take bacteria as food and multiply as a unicellular organism under favorable conditions. Upon starvation, a pathway consists of aggregation, mound, slug, and culmination stages induces the formation of a fruiting body which has a head of spores supported on a stalk of vacuolated cells. Spores await dispersal and germinate in favourable conditions for amoeboid growth. The genome of Dictyostelium discoideum is haploid, that is a major advantage compared with other model systems because it can generate a rich variety of mutants. The multi-cellular development process is relatively short (24h) and the regulatory signaling pathways are familiar with those in metazoan development, Dictyostelium discoideum is an excellent system in which to study cell mobility, cell-cell signal transduction, cell type differentiation, development and so on.Gp150 which encoded by lagC gene is a membrane glycoprotein and plays an important role in regulating cell differentiation and multicellular development. The mutant type strain AK127, which the lagC gene is absent, can’t complete multicellular development and the development arrests at the aggregate stage. This suggested that gp150 protein is very important to the development of Dictyostelium discoideum. Distribution of gp150 is different in prestalk cells and prespore cells. Gp150 mainly exists in prestalk cells, while there is nearly no gp150 existing in prespore cells. Adhesion molecules are the candidates participating in intra- and inter- cellular signal transduction. So far, our knowledge of gp150’s signaling pathway is still very limited.In order to screen the interacting proteins of gp150, protein gp150 (lagC C-terminal) was expressed and polyclonal antibody of gp150 C-terminal was prepared and identified. The recombinant plasmid pET-32a(+) with lagC C-terminal and 6 His tagged was expressed in E. coli BL21 (DE3) host cells. The fusion protein with 6xHis was purified by Ni2+ affinity chromatography column and then was used to immune rabbits for preparing polyclonal antibody. The titer and specificity of the rabbit’s antiserum were measured by ELISA and Western blotting, respectively. The results showed that the fusion protein was successfully expressed and polyclonal antibody was also successfully obtained. The specificity of antibody was proved by Western blotting analysis of expression product of gp150. The potency of the polyclonal antibody was as high as 1:128000. The polyclonal antibody was used in Co-immunoprecipitation and seven gp150-intertacting proteins were captured, whose molecular weight is from 30KD to 15KD.We choose two clear protein bands for identifing by mass spectrometer.The result shows that the two protein is 40S ribosomal protein S3 and 40S ribosomal protein S24, which play important roles in regulation of cell cycle, cell development and apoptosis. The data suggests that gp150 can interact with RPS3 and RPS24 for regulating cell differentiation and development during multicellular development of Dictyostelium discoideum.This research lays the foundation for the study of gp150’s role in the signal conduction of Dictyostelium discoideum. It also contributes to the study of the whole signal pathway of Dictyostelium discoideum.更多还原