噬菌体phiC31整合酶在牛基因组中介导基因定点整合的分子机制研究

The Streptomyces phage phiC31 integrase can effectively integrate attB-containing transgenes directly into mammalian genomes at endogenous pseudo attP sites. It is a potential tool for both gene therapy and generation of transgenic animals,however,the mechanism of site-specific integration is not clear. In our previous study, 32 integration sites were found in bovine genome for the first time and by analyzing the DNA sequences of pseudo attP sites, we found that DNA sequence recognition might play an important role in guiding phiC31-mediated site-specific integration. In this study, we will establish a bovine cell line stably expressing phiC31 integrase protein, use chromatin immunoprecipitation- sequencing(ChIP-seq) method to find the DNA sequences phiC31 integrase binding, analyze the sequence characters of pseudo attP sites and the sequence around them by bioinformatics methods. We will also analyze the influence of histone acetylation on site-specific integration by phiC31 integrase,in order to find if the epigenetic modification plays a role in regulating the function of this integrase in bovine genome. In this project, we study the action characters of phiC31 integrase on the genome scale for the first time, analyze the mechanism of this system from the aspects of sequence recognition and epigenetic modification.The research will accelerate the application of phiC31 integrase in genetic engineering, and will provide theoretical support for improving the efficiency and safety in the field of gene transfer and gene therapy.

噬菌体phiC31整合酶介导外源基因在基因组中的特异性整合是转基因生物和基因治疗领域最有希望的方法之一，但其在真核细胞中介导定点整合的机制研究还不深入。我们在前期工作中首次鉴定了牛基因组中32个整合酶位点，发现DNA序列可能是整合酶发挥定点整合作用的重要影响因素。本研究拟在此基础上构建表达phiC31整合酶的牛细胞株，通过染色质免疫共沉淀-测序技术分析phiC31整合酶在基因组范围内识别结合的DNA序列；运用生物信息学方法分析其作用位点及旁侧序列的序列特征；通过研究组蛋白乙酰化状态改变对整合酶识别位点的影响，来探讨表观修饰在引导整合酶对位点特异性识别上所发挥的作用。本项目首次在全基因组范围、从序列特征和表观修饰两方面来研究整合酶在牛基因组中介导定点整合的机制，这些研究将为加快phiC31整合酶在基因工程中的应用、为提高转基因操作和基因治疗的安全性有和效性提供理论支持。

噬菌体phiC31整合酶能介导外源基因定点整合于哺乳动物基因组中的特征在生命科学领域具有重要的应用价值，但是其介导基因定点整合的机制研究还不深入。在前期的研究中，我们发现phiC31整合酶可以在牛基因组中发挥定点整合的作用，并鉴定了32个假attP位点。本次研究中，我们构建表达phiC31整合酶蛋白的真核表达载体，稳定转染的方法制备表达整合酶蛋白的转基因MDBK细胞，并通过测序及蛋白电泳等方法来鉴定细胞构建与否。通过染色质免疫共沉淀的方法，捕获phiC31整合酶蛋白在牛基因组中作用结合的DNA片段。对捕获的片对，通过检测PCR及定量PCR的方法，来鉴定捕获是否成功。将捕获的片段，经过高通量测序的方法，鉴定出6740富集片段，总长度4,136,812，平均613.77。通过MEME-motif分析，发现4个整合酶结合motif，并且这些motif呈串联重复排列特点。通过GO及KEGG功能注释，分析整合酶结合位置相关基因的功能；通过REPEATMASKER软件分析富集片段附近重复序列分析发现，整合酶作用位点上及位点的上下游序列中存在大量的重复序列。这些位点及序列特征很好的解释了phiC31整合酶特异性位点结合的分子机制，为更好地利用、改造整合酶提供理论基础。