中文摘要：

【目的】探明Cdc42蛋白在牛卵母细胞成熟过程中的表达及分布规律，并且研究抑制Cdc42活性对于牛卵母细胞成熟的影响。【方法】首先通过收集体外成熟不同时间（0—24 h）的牛卵母细胞，用Western blotting的方法检测Cdc42蛋白在牛卵母细胞中的表达量变化情况，通过免疫细胞化学的方法检测Cdc42蛋白在牛卵母细胞中的分布及定位情况。根据GenBank公布的牛Cdc42基因（GenBank登录号：NM_001046332.1）的mRNA序列设计引物，并在正向和反向引物的末端分别加上KpnⅠ和EcoRⅠ酶切位点，用PCR的方法扩增牛Cdc42基因的CDS区，将PCR产物和pMD19T载体连接得到野生型的克隆载体pMD19T-Cdc42WT。用定点突变引物以pMD19T-Cdc42WT为模板进行PCR反应，得到Cdc42的显性负性突变体Cdc42T17N的克隆载体pMD19T-Cdc42T17N。将pMD19T-Cdc42WT和pMD19T-Cdc42T17N分别酶切后，将Cdc42WT（775 bp）和Cdc42T17N（775 bp）片段分别连接到pcDNA3.1（＋）的多克隆位点区域，得到相应的原核表达载体pcDNA3.1（＋）-Cdc42WT和pcDNA3.1（＋）-Cdc42T17N。用体外转录的方法分别得到Cdc42WT和Cdc42T17N的cRNA，用显微注射的方法将相应的cRNA分别注入到牛GV期卵母细胞质内，检测其成熟率的变化。【结果】①Cdc42蛋白分别在牛卵母细胞中成熟培养0、4、8、12、16、20以及24 h时表达量没有明显差异，但是其分布规律随着减数分裂的进行发生着动态的变化：Cdc42蛋白在卵母细胞皮质区域集中分布的模式在生发泡（germinal vesicle，GV）期卵母细胞中很少出现，而在第一次减数分裂中期（metaphase Ⅰ，MⅠ）卵母细胞中出现得最多也最为明显；Cdc42蛋白在染色体附近的皮质富集的现象开始出现于前中期（pro-metaphase Ⅰ，pMⅠ），并且出现频率从pMⅠ期到第2次减数分裂中期（metaphase Ⅱ，MⅡ）逐渐增多；Cdc42蛋白和纺锤体重叠定位的现象在第1次减数分裂后?

英文摘要：

【Objective】 This study is designed to ascertain the expression and localization of Cdc42 protein during bovine oocyte maturation, and to clarify the effect of Cdc42 inhibition on maturation of bovine oocytes. 【Method】 Bovine oocytes in vitro matured for different times （0-24 h） were collected. Western blotting and immunocytochemical technique were adopted to detect the expression and distribution of Cdc42 protein from oocytes collected at different stages of in vitro maturation. A pair of primers with restriction enzyme sites of KpnⅠand EcoRⅠat the ends of forward primer and reverse primer respectively, was designed based on the mRNA sequence of bovine Cdc42 which was obtained from Genbank （accession No.： NM_001046332.1）. The CDS of bovine Cdc42 gene was amplified by PCR, and then ligated with pMD19T vector to construct pMD19T-Cdc42WT, the cloning vector wild type of Cdc42. A pair of site-directed mutagenic primers was used in a PCR to obtain pMD19T-Cdc42T17N, the cloning vector of the dominant negative mutant of Cdc42. Both pMD19T-Cdc42WT and pMD19T-Cdc42T17N were digested by restriction endonuclease to obtain the fragment Cdc42WT （775 bp） or Cdc42T17N （775 bp）. The obtained fragments were ligated to the multiple cloning site of pcDNA3.1（＋） to construct the prokaryotic expression vectors. The cRNA of Cdc42WT and Cdc42T17Nwas synthesized by in vitro transcription, and then micro injected into the cytoplasm of bovine GV stage oocytes. The maturation rate of injected oocytes was detected. 【Result】 The expression level of Cdc42 protein remained constant over the whole bovine oocytes maturation process, but the distribution pattern varied as the meiosis proceeded. The distribution pattern that Cdc42 protein gathering at the cortex region was hardly found in oocytes of germinal vesicle （GV） stage, but occurred frequently and remarkably in metaphase Ⅰ （MⅠ） oocytes. The distribution pattern that Cdc42 protein gathering at the cortex region nearing meiosis apparatus rat