嗜肺军团菌血清10型信号标签诱变突变体文库构建

发布时间：2018-05-08 08:55

本文选题：信号标签诱变 + 噬肺军团菌　；参考：《四川大学》2006年硕士论文

【摘要】：目的：本文通过PCR扩增特异信号标签，导入载体质粒pC6，构建信号标签诱变突变体文库穿梭质粒pC6RS；通过细菌结合配对时转座子的转座作用，，使特异信号标签随机插入到嗜肺军团菌的基因组中，由此构建嗜肺军团菌血清10型信号标签诱变突变体文库，为筛选嗜肺军团菌血清10型毒力基因打下基础。包括二部分：1、信号标签诱变突变体文库穿梭质粒构建；2、嗜肺军团菌血清10型信号标签诱变突变体文库的建立。方法：实验一：信号标签诱变突变体文库穿梭质粒构建通过PCR扩增信号标签(signature tags，STs)片段，导入载体质粒pC6，构建重组质粒pC6RS，电穿孔转化到大肠杆菌CC118中，从CC118中提取重组质粒pC6RS后转化到大肠杆菌S17-1中，获得信号标签诱变突变体文库穿梭质粒。实验二：嗜肺军团菌血清10型信号标签诱变突变体文库的建立含重组质粒pC6RS的大肠杆菌S17-1与嗜肺军团茵L10结合配对，通过转座子的转座作用，将特意信号标签片段随机插入到军团菌的基因组中，通过营养依赖和抗性筛选建立嗜肺军团菌血清10型信号标签诱变突变体文库。结果：实验一：信号标签诱变突变体文库穿梭质粒构建通过PCR扩增信号标签(signature tags，STs)片段，导入载体pC6，构建重组质粒pC6RS，电穿孔转化
[Abstract]:Objective: In this paper, the specific signal tags were amplified by PCR and introduced into vector plasmid pC6. the shuttle plasmid pC6RSwas constructed by mutagenesis of signal label, and the transposon of transposon was matched by bacterial binding. The specific signal label was inserted into the genome of Legionella pneumophila at random, and the mutant library of serotype 10 signal label was constructed, which laid the foundation for screening the virulence gene of serotype 10 of Legionella pneumophila. It includes two parts: 1, shuttle plasmid construction of signal label mutagenesis mutant library, and establishment of the library of Legionella pneumophila serotype 10 signal label mutagenesis mutant library. Methods: Experiment 1: the shuttle plasmid of signal label mutagenesis mutant library was constructed by PCR amplification of signature tag STs fragment, then introduced into vector plasmid pC6, constructed recombinant plasmid pC6RSs, electroporated into Escherichia coli CC118. The recombinant plasmid pC6RS was extracted from CC118 and transformed into Escherichia coli S17-1. The shuttle plasmid of signal label mutagenesis mutant library was obtained. The second experiment: the establishment of mutagenesis library of Legionella pneumophila serotype 10 signal label. Escherichia coli S17-1 containing recombinant plasmid pC6RS was paired with Legionella pneumophila L10, and transposon was used. The special signal tag fragment was randomly inserted into the genome of Legionella pneumophila, and the mutant library of Legionella pneumophila serotype 10 signal label mutagenesis was established by nutritional dependence and resistance screening. Results: Experiment 1: signal label mutagenesis mutant library shuttle plasmid was constructed by PCR amplification of signal tag signature tagsfest STs fragment, introduced into vector pC6, constructed recombinant plasmid pC6RSs, electroporation transformation
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R378

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