鼠疫菌中国强毒株（910706）全基因组BAC文库的构建

发布时间：2018-01-05 13:05

本文关键词：鼠疫菌中国强毒株（910706）全基因组BAC文库的构建　出处：《中国人民解放军军事医学科学院》2009年硕士论文　论文类型：学位论文

【摘要】： 背景鼠疫是一种以鼠疫耶尔森氏菌为病原菌的烈性传染病,目前,鼠疫的致病机制,流行机制还没有被人类所完全认识。鼠疫菌基因组进化过程经历了大量的遗传物质流动,通过基因水平转移基因重组移码突变和点突变获得大量质粒和染色体基因,产生了大片段染色体片段重排,积累了大量假基因。各个分型的染色体中各有特有的基因组片段,导致了各分型间的基因组结构和功能的差异,针对这些差异和机制所进行的基因组学和比较基因组学研究,将是今后鼠疫菌研究的一个方向。基因组文库技术是一种能够对基因组进行纯化、贮存并在此基础上进行广泛深入研究的分子生物学技术,其中,细菌人工染色体(BAC)是20世纪90年代发展起来的新型DNA文库载体技术,具有稳定性好,不易发生缺失重排,少有载体中毒效应等优点,在染色体的组织进化基因的组织结构分析特定染色体基因的克隆基因组组织和基因表达基因组物理图谱的构建以及基因组测序等基因组研究的多个领域,BAC文库都获得了广泛的应用。方法通过提取、纯化得到鼠疫菌基因组DNA,经限制性内切酶部分酶切后,与经过末端去磷酸化处理的CopyControlTMpCC1BACTM Clone-ready载体按照一定摩尔比例相互连接,转化TransforMax EPI300并涂板,经蓝白斑筛选,筛选阳性克隆。通过分析阳性克隆的末端测序结果,确认克隆中插入片段中的来源。分别提取不同传代的BAC克隆的质粒DNA,用Hind III酶切分析比较指纹图谱的变化,分析BAC克隆在继代培养中是否稳定。从所建库中随机挑选克隆,小量提取质粒后脉冲电泳鉴定,以计算插入片段的平均长度,空载率和覆盖率。结果末端测序及指纹图谱分析结果,证明所建BAC文库中插入片段确来源于宿主菌,且稳定性良好。通过脉冲电泳及相关试验结果计算,该库共包含3248个已测序克隆,平均插入片段为105 kb空载率为2%总的库容为35Mb,约8倍于鼠疫菌基因组覆盖率为97%。鼠疫强毒株BAC文库构建成功。
[Abstract]:The background is a plague plague Jerson Prand for pathogens of infectious diseases, the pathogenesis of plague, epidemic mechanism has not been fully recognized. The human plague bacteria genome evolution experienced a lot of genetic material flow through horizontal gene transfer of recombinant gene?? frameshift and point mutations and get a lot of plasmid the chromosomal gene, produced a large fragment of chromosome rearrangement, the accumulation of a large number of pseudogenes. A unique genomic fragment of each type of chromosome, lead to differences in the structure and function of the genome of each type of Inter, for these differences and the mechanism of genomics and comparative genomics research, will be a the research direction in the future. Yersinia pestis genome library technology is a kind of the genome of the purification, storage and on the basis of molecular biology and extensive research The technology of bacterial artificial chromosome (BAC) is a new type of DNA library vector technology developed in 1990s, with good stability, less prone to lack of rearrangement, little carrier poisoning effect etc, in the evolution of organization structure analysis of chromosome? Gene cloning? Chromosome specific gene expression gene and genome organization? How? A study in the field of construction and genome sequencing genome physical map, BAC library is widely used.
Through the method of extraction, purification of Y.pestis genomic DNA by restriction endonuclease digestion and CopyControlTMpCC1BACTM part, the Clone-ready vector after the end of dephosphorylation according to certain molar ratio of TransforMax and EPI300 are connected to each other, conversion coated plate, by blue white screening, the positive clones were screened by sequencing. Results the positive clones confirmed. Source of the clones. Plasmid DNA were extracted from different BAC clones were the changes of fingerprint comparison by Hind III enzyme digestion and analysis of BAC clone in the following Daipei in raising the stability. From the database of randomly selected clones, PFGE plasmid miniprep, to calculate the average length of inserted fragments the load rate and the coverage rate.
The results of end sequencing and fingerprint analysis results show that the proposed BAC library inserts is derived from the host bacteria, and good stability. By PFGE and related experimental results, the library contains a total of 3248 sequenced clones with an average insert size of 105 KB? The no-load rate was 2%? The total capacity is 35Mb. About 8 times in the Y.pestis genome? Coverage for 97%. Plague Virulent Strain BAC library was successfully constructed.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R378

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