【作者】 孙难民；

【导师】 田海林；

【作者基本信息】 苏州大学 ， 劳动卫生与环境卫生学， 2009， 硕士

【摘要】 目的:应用特殊设计的寡核苷酸链纠正点突变这一技术是近年发展起来的一项分子生物学新技术,应用该技术能在基因组碱基序列中任意位点纠正或引入一个指定的碱基。较之传统的基因重组技术,该技术对基因组本身的结构几乎没有任何影响,且不会产生因病毒载体和质粒载体引起的免疫反应以及随机整合。一些遗传性疾病,如Duchenne肌营养不良症(DMD),有部分是抗肌萎缩蛋白基因的点突变引起的。本研究旨在建立应用特殊设计的单链寡核苷酸链纠正点突变这一新技术的研究方法,建立新的试验体系,探索不同设计的单链寡核苷酸链(ODN)对点突变的纠正率,筛选出更佳设计的ODN及确定最佳ODN浓度。并将该技术初步应用于纠正抗肌萎缩蛋白基因的点突变,为今后将该技术应用于DMD等由基因点突变而引起的疾病的基因治疗提供实验依据。方法:本研究分为三部分,第一部分为以Ames试验体系为基础,探索使用特殊设计的单链寡核苷酸链(ODN)纠正TA102中组氨酸操纵子基因的点突变的试验方法,建立新的试验体系。先用Ames试验检测ODN对细菌突变纠正能力,然后探索在细菌快速增长期加入的ODN对细菌的突变纠正情况,最后用CaCl2将细菌制备成感受态细菌后,研究ODN对细菌的突变纠正能力。第二部分为使用长度分别为31、45、60、75个碱基的模板链ODN和31个碱基的编码链ODN纠正TA102中组氨酸操纵子基因的点突变,使用不同浓度的ODN纠正该基因的点突变,筛选出更佳设计的ODN及确定最佳ODN浓度。第三部分为初步探讨使用特殊设计的单链寡核苷酸链(ODN)纠正抗肌萎缩蛋白基因的点突变,给予DMDmdx小鼠肌肉注射低剂量和高剂量的ODN,以及另外附加透明质酸酶预处理和活体电穿孔术,采用扩增阻滞突变体系(ARMS)法检测点突变纠正情况。结果:将ODN直接加入到正常状态下的细菌中检测不出突变纠正率,而用CaCl2法将TA102制备成感受态细菌后可以得到一定的纠正率(10-5)。实验使用的这几个长度的ODN纠正TA102中组氨酸操纵子基因的点突变效率均很低,长度为75bp的ODN纠正效率相对高一些;模板链ODN比编码链ODN纠正率高;实验使用的几个浓度中浓度为5ng/μL的纠正率相对高些。而体内实验结果显示,无论低剂量或高剂量ODN肌肉注射,还是在此基础上另外再采用透明质酸酶预处理和电穿孔术,都检测不到纠正了的基因条带。结论:ODN能够纠正TA102中组氨酸操纵子基因的点突变,可以建立起以Ames试验体系为基础的研究寡核苷酸链纠正点突变的实验方法。ODN纠正TA102中组氨酸操纵子基因的点突变存在链偏性,即作用于非转录链的模板链ODN比作于转录链的编码链ODN效率高;长度为75bp的模板链ODN对该基因的纠正效率相对高一些,最佳ODN浓度为5ng/μL。体内试验中,所使用的实验方法不能纠正抗肌萎缩蛋白基因的点突变或因突变纠正率过低ARMS法检测不出。更多还原

【Abstract】 Objective: The technique that correcting the point mutation by specially designed oligonucleotides(ONs) is a latest molecular biological technique developed in recent years. With this technique, we can correct or introduce a assigned point mutation in any site of genes, which sequence already known. Contrast to the traditional gene recombination method, this technique hasn’t any influence on the construction of genome itself and doesn’t produce immunoreaction and random integration caused by virus or plasmid vectors. Some genetic diseases, such as Duchenne muscular dystrophy (DMD), are partly caused by a given point mutation. In this study, we aim to establish a new empirical method for studying oligonucleotide-mediated gene repair of point mutation. We tested the ability of the different designed single-stranded oligonucleotides (ODNs) to correct the point mutation, determined the better structure and the optimal concentration of ODN. In vivo studies,we had tried to correct the point mutation of dystrophin gene by specially designed single-stranded oligonucleotides and provided some experimental data for this technique to be ultimately used in gene therapy of DMD or other diseases caused by point mutation.Methods: This study was divided into three parts. In the first part, basing on Ames test system, we had tried to establish a new empirical method in which we used ODN to repair the point mutation in the gene of histidine operon of TA102. First, we tested ODN’s ability to correct the point mutation by Ames test; then explored the correcting state when ODN mixed in bacterium which was in the period of rapid growth and,at last,studied ODN’s ability to correct the point mutations when it mixed in competence bacterium which prepared by CaCl2. In the second part, several different concentrations of ODN and different structures of ODNs were performed to correct the point mutation in the gene of histidine operon of TA102.The different ODNs including 31、45、60、75bp length of template strands and 31bp length of coding strands were used. In vivo studies we administrated low does and high does ODN to DMDmdx mouse by intramuscular injection, or pretreated with hyaluronidase and did electroporation in vivo in addition. We tried to detect the corrected gene by amplification refractory mutation system (ARMS) assay.Conclusion: ODN can repair the point mutation in the gene of histidine operon of TA102 and the empirical method which baseing on Ames test system used for studying gene repair of point mutation by ODN is available. In this new system, strand bias is exist when using ODN to repair the point mutation in the gene of histidine operon of TA102; the 75bp length of template strand has a better repair efficient relatively, and 5ng/uL is the best concentration. But in vivo studies,what we used methods cannot correct the point mutation of dystrophin gene in vivo, or the repair efficiency was too low to be detect by ARMS assay.更多还原