人AAVS1位点的TALENS质粒构建及活性验证

摘要：本实验旨在利用类转录激活子样效应因子核酸酶（transcription activator -like (TAL) effector nucleases TALENS）技术构建具有活性的AAVS1的TALEN质粒。根据TALEN设计原则和AAVS1的共同序列确定基因敲除的靶位点、TALEN识别序列和用于活性验证的限制性酶切位点。利用质粒文库快速构建TALEN质粒。通过氯化钙法将质粒共转入293T细胞，同时加入两种常见的质粒转染增强试剂氯喹和丁酸钠, 以对比不同药物对TALEN质粒活性的影响。结果显示：两种质粒转染增强药物对TALEN质粒活性的影响不显著，推测原因一是通过T7E1酶切验证活性不是一种很好的定量方法，二是未使用增强酶切活性的最佳药物剂量及作用时间。36506
毕业论文关键词：TALEN质粒；AAVS1；活性验证；293T；氯喹；丁酸钠
Construction and Activity Assay of Transcription Activator—like Effector Nuclease(TALEN)Plasmids for AAVS1
Abstract：The present study is designed to take advantage of transcription activator-like effector nucleases (transcription activator-like (TAL) effector nucleases TALENS) to constructing TALEN plasmid having the activity AAVS1. According to the common sequence TALEN design principles to determine the target site and AAVS1 knockout, TALEN recognition sequence and for active authentication restriction sites. Using plasmid library kit quickly build TALEN plasmid.By Calcium chloride method plasmids into 293T cells and add chloroquine and sodium butyrate to compare the effects of different drugs on the activity of the plasmid TALEN.The results showed that two plasmid vector to enhance the impact of drugs on TALEN activity was not significant. One reason speculation verified by T7E1 enzyme activity is not a good quantitative method, the second is not used to enhance the activity of enzyme optimal drug dose and duration of action.
Keywords: TALEN plasmid AAVS1 Activity Assay 293T Chloroquine Sodium butyrate
目录
1 绪论    - 1 -
1.1 本课题的国内外研究进展    - 1 -
1.1.1 TALENS    - 1 -
1.2 AAVS1 位点    - 2 -
1.2 本课题的研究内容、目的及意义    - 3 -
1.2.1 TALENS    - 3 -
1.2.2 AAVS1    - 3 -
2 材料与方法    - 5 -
2.1 实验材料    - 5 -
2.1.1 TALENS质粒构建    - 5 -
2.1.2活性验证    - 5 -
2.2 实验方法    - 5 -
2.2.1 TALENS质粒构建实验流程：    - 5 -
（1）人AAVS1 在基因组中的序列信息：    - 6 -
(2) 目的基因扩增    - 7 -
(3) PCR 扩增起始片段    - 8 -
(4) 用BbsI和BsaI-HF酶切获得延伸片段和载体片段    - 8 -
(5) 准备预混合液    - 9 -
(6) 磁珠的准备    - 9 -
(7) 全长片段的制备    - 9 -
(8)表达载体线性化    - 10 -
(9)全长片段和线性化载体的连接    - 10 -
(10) 转化    - 11 -
(11) 阳性克隆鉴定    - 11 -
(12) 质粒抽提    - 11 -
2.2.2 活性验证    - 11 -
(1 )TALENs细胞样品的准备：    - 11 -
(2) PCR    - 12 -
(3) T7E1活性分析实验-退火    - 12 -
(4) T7E1活性分析实验-酶切    - 12 -
(5) T7E1活性分析实验-DNA PAGE电泳，染色    - 12 -
3 结果与讨论    - 13 -
3.1起始片段的制备结果    - 13 -
3.2 延伸片段和终止片段制备结果    - 13 -
3.3.菌落PCR鉴定阳性克隆    - 14 - 人AAVS1位点的TALENS质粒构建及活性验证:http://www.youerw.com/shengwu/lunwen_34992.html