原生动物八肋游仆虫cDNA文库的构建

Construction of cDNA library of protozoan ciliates Euplotes octocarinatus

细胞内蛋白质合成过程是一个由多种蛋白质相互作用参与调节的开放系统,形成了复杂的mRNA代谢和蛋白质翻译为核心的基因表达调控的网络和信号转导途径。【目的】为了获得更多参与调节蛋白质合成终止过程的蛋白质种类和功能信息,进一步了解其中的网络和信号转导途径,本研究构建了原生动物八肋游仆虫的cDNA文库。【方法】构建过程严格遵循Clontech公司的BD MatchmakerTM Library Construction&Screening kit提供的方案进行文库构建和筛选.【结果】首次得到了可用于筛选功能基因的原生动物纤毛虫的cDNA文库,文库滴度为2.437×107cfu/mL。利用第二类肽链释放因子为诱饵,筛选得到了一些可能与之相互作用的蛋白质,其中包括一个可能编码RNA解旋酶的基因序列。该文库为进一步筛选和研究八肋游仆虫功能基因提供了便利的平台。

The protein biosynthesis in cells is an open process cooperatively regulated by many protein factors and enzymes, which form a complicated protein network and signal transduction pathway for mRNA metabolism and protein translation. [Objective]To provide a platform for studying on the function of proteins involving in process of translation termination and the relationship among these proteins in ciliates. [Methods] We constructed the cDNA library of protozoan ciliates Euplotes octocarinatus strictly following the procedure of BD Matchmaker^TM Library Construction Screening kit from Clontech. [Results] We obtained a cDNA library of ciliates Euplotes, the titer of which was about 2.437×10^7 cfu/mL, which could suffice for functional gene screen. By using the class II polypeptide release factor （eRF3） as bait protein, we obtained some putative genes, including a partial cDNA putatively encoding RNA helicase. [Conclusion] This library will provide a convenient platform for identifying the functional genes in ciliates.