摘要
目的探索卵巢成熟及明确早胚发育过程中,构建小鼠卵母细胞cDNA文库筛选所涉及的相互作用蛋白。方法利用SMART技术,构建酵母双杂交筛选卵母细胞中与输卵管蛋白相互作用的靶分子成分为目的,构建小鼠卵母细胞cDNA酵母双杂交文库。采用Stratagene RNA prepkit抽提500枚卵母细胞总RNA,应用线性化载体pGADT7-Rec在体构建小鼠卵母细胞cDNA酵母双杂交文库。结果文库的滴度为3.7×106,重组片段大小主要集中在0.6-0.9kb,重组率为60%。结论所构建的小鼠卵母细胞cDNA文库可用于酵母双杂交筛选。
Purpose In order to identify the network and the components involved in the follicular development and early embryogenesis, a cDNA library from mouse oocyte for yeast-2-hybrid system was to be constructed by homologous recombination to screen early embryo development-related gene. Methods A PCR-based cDNA amplification method （Smart technology） was used. Total RNA of 500 mouse oocytes was extracted with the one step method of RNA by acid guanidinium thiocyanate-phenol-chloroform extraction with Stratrgene RNApre kit. The double -stranded cDNA was synthesized using MATCHMAKER kit. The Sma I-linearized PGADT7-rec was cotransfected to Saccharomyces cerevisiae AH109 with the amplified double -stranded cDNA. Results The titer of the amplified cD- NA library was 3.7 × 10^6 cfu/ml,the inserted fragment size of recombinants from 0.6-0.9 kb and the percentage recombinant clones was 60%. Conclusions The results determined was that this mouse oocyte cDNA libraries was beneficial for yeast-2-hybrid screening.
出处
《复旦学报（医学版）》
CAS
CSCD
北大核心
2007年第4期599-602,共4页
Fudan University Journal of Medical Sciences
基金
国家"973"项目(G1999055902)
