DNA复制是细胞生命进程中不可或缺的一部分，它的精确完成是生命体繁殖及细胞增殖的基础。DNA复制进程依赖于多种蛋白，其中Cdc45/Mcm2-7/GINS（CMG）复合体是复制过程中的解旋酶，它能将双链DNA解旋为单链，从而为DNA聚合酶提供模板底物。CMG复合体在S期早期组装、激活并与相关蛋白共同进行DNA复制。当细胞遭遇复制压力时，S期细胞周期检验点ATR-CHK1通路将被激活且CMG复合体的功能可能将被细胞周期检验点调节，然而其具体机制还未得到阐明。因此，为探究细胞周期检验点调控CMG复合体的机制，我们需要在体外环境下获得完整的CMG复合体。为此，本研究构建了高表达CMG复合体的质粒，并将其定点插入至裂殖酵母细胞基因组中的特定位点，进而获得高表达CMG蛋白复合物的稳定菌株。而后，通过串联亲和层析的方法依次纯化并富集Cdc45和GINS蛋白的相互作用蛋白，并通过蛋白质免疫印迹法和串联质谱分析鉴定纯化的组分是否为完整的CMG复合物。实验结果表明，完整的CMG复合体能够在重组菌株中稳定表达并在体外环境下成功纯化，以此为后续CMG复合体在S期细胞周期检验点相关通路中的研究提供帮助。

DNA replication is an essential part of basic process in cell. Its accuracy influences life propagation and cell proliferation fundamentally. DNA replication relies on several proteins, one of them is the replicative helicase called Cdc45/Mcm2-7/GINS (CMG) Complex. It unwinds double-strand DNA to single-strand DNA, and thereby provides DNA polymerase with substrate. CMG Complex is assembled and activated and then replicates DNA with other proteins in S phase. Once cells are under replication stress, S-phase-checkpoint ATR-CHK1 pathway is activated, leading to the functional regulation of CMG Complex. However, the mechanism is still unclear. In order to figure out the mechanism in this regulation, we need to get the whole CMG Complex in vitro. Therefore, in this experiment, over-expressed plasmids were constructed, inserted into the specific site of fission yeast genome and then we obtained the over-expressed strains of CMG Complex. After that, Tandem Affinity Purification was used to purify and enrich the proteins which interact with Cdc45 and GINS, then we identified the component whether it is the whole CMG Complex by Western Blot and Tandem Mass Spectrometry. The results show the whole CMG Complex is able to be successfully over-expressed in recombinant strains and purified in vitro. Then it would become the material of the research on CMG Complex in S-phase-checkpoint pathway.