【作者】 张小男；

【导师】 陈珊； 陈惠鹏；

【作者基本信息】 东北师范大学 ， 微生物， 2010， 硕士

【摘要】 基因表达的调控是分子生物学研究的一个重要问题。特别是在转录水平,其调控是一个非常复杂的过程。目前存在的一些问题给基因的表达和调控研究造成了困难,如一些基因的持续表达则接近细胞毒水平会造成细胞的死亡、毒性基因功能研究缺少精准的调控系统,以及以生产毒性蛋白为目标的工程化细胞株的构建难等问题。因此构建一种严谨、高效以及可诱导调控的基因表达调控系统,可人为控制外源基因的表达水平,有利于模拟该基因在生理、病理等不同状态下的作用特点和机制。利用这种可诱导的调控系统可以用于定量调控基因表达以及进行毒性基因功能分析等方面的研究。目前所有的真核基因表达调控系统中四环素调控系统(tet systerm)被认为是目前最为理想的真核生物基因表达调控系统。其中四环素调控系统中tTS/tet-on调控系统应用最为广泛,该系统的组成为:两个调控蛋白(tTS,rtTA)和一个四环素反应表达框(TRE),该系统的诱导物为四环素(Tc)或其衍生物强力霉素(Dox)。两种调控蛋白的联合使用不仅不会影响该系统的诱导能力,对其基础表达水平也能大大降低,现已经广泛应用于构建各种细胞模型以研究目的基因的功能之中。本研究利用新型tTS/tet-on调控表达载体,选择CHO-K1为宿主细胞,构建并筛选出稳定的tTS/tet-on细胞株。通过Real-time PCR和双萤光素酶报告基因检测系统对筛选出的tTS/tet-on调控表达细胞株的调控性能进行测定和比较。Real-time PCR结果及萤光素酶测定数据表明,筛选出的tTS/tet-on调控表达细胞株更为严谨和高效,取得了较好的实验结果。在此基础之上,利用筛选出的tTS/tet-on调控表达细胞株,进行病毒2A蛋白酶基因的表达调控研究,证明严谨型tTS/tet-on调控表达细胞株可以实现对毒性蛋白的调控表达。本研究成功构建并筛选出两株严谨型tTS/tet-on调控表达细胞株,其较低的基础表达活性及较强的诱导能力为进行外源性兴趣基因在真核细胞中的调控表达提供了一个技术平台。更多还原

【Abstract】 Gene expression and regulation is an important area in Molecular Biology, particularly in the level of transcription, its regulation is a very complex process.At present some problems caused difficulties for gene expression and regulation.Such as the continued expression of certain genes are toxic to cells and cause cell death, the lack of accurate system to study virulence gene function,and the production of toxic proteins targeted cell line engineering construction,etc.To construct a tight, effective and inducible gene regulatory expression system for human-controlled gene expression levels,it conducive to simulate the gene in physiological and pathological roles of different state characteristics and mechanisms.Using this inducible system can be used for quantitative regulation of gene expression regulation and virulence gene function analysis and so on.Tetracycline control system (tet systerm) is regarded as the most ideal eukaryotic gene expression system in all eukaryotic gene regulatory exression system.Nowadays the most frequently and widely used eukaryotic gene regulatory expression system is the tTS/tet-on system of tet systems. This regulatory system consisted of: two regulatory proteins(tTS,rtTA), and a response element(TRE),inducer of the system is the tetracycline (Tc) or its derivatives doxycycline (Dox).The joint use of two kinds of regulatory proteins are not only will affect the ability to induce,but also the basis of their expression levels can be greatly reduced.Now it has been widely used in building a variety of cell models to study gene function.In this study, according to a new building completed tTS/tet-on regulation of expression vector,choose CHO-K1 as the host cell to build and filt stable tTS/tet-on cell line.By Real-time PCR and firefly luciferase as the reporter to have the constructed and filted tTS/tet-on cell lines been compared in the field of regulation capabilities. The data of Real-time PCR anf firefly luciferase indicate that screened tTS/tet-on cell lines with lower expression level and more effective,achieved a good results.In this study,we also try to regulate the synthesis of toxic protein 2Apro by screened this new type of tTS/tet-on gene regulatory expression cells by preliminary discussion the results,the tight type tTS/tet-on cell lines can be achieved on the expression and regulation of toxic protein.This study has successfully constructed and screened two tTS/tet-on gene regulatory expression cells.Its lower expression level and high inductive ability could provides a technology platform for the tightly regulatory expression of.exogenous protein in eukaryotic cells.更多还原