本文關(guān)鍵詞：鼠疫菌中國(guó)強(qiáng)毒株（910706）全基因組BAC文庫(kù)的構(gòu)建　出處：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2009年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 鼠疫耶爾森氏菌 強(qiáng)毒株 基因組 BAC文庫(kù)

【摘要】： 背景鼠疫是一種以鼠疫耶爾森氏菌為病原菌的烈性傳染病,目前,鼠疫的致病機(jī)制,流行機(jī)制還沒(méi)有被人類(lèi)所完全認(rèn)識(shí)。鼠疫菌基因組進(jìn)化過(guò)程經(jīng)歷了大量的遺傳物質(zhì)流動(dòng),通過(guò)基因水平轉(zhuǎn)移基因重組移碼突變和點(diǎn)突變獲得大量質(zhì)粒和染色體基因,產(chǎn)生了大片段染色體片段重排,積累了大量假基因。各個(gè)分型的染色體中各有特有的基因組片段,導(dǎo)致了各分型間的基因組結(jié)構(gòu)和功能的差異,針對(duì)這些差異和機(jī)制所進(jìn)行的基因組學(xué)和比較基因組學(xué)研究,將是今后鼠疫菌研究的一個(gè)方向�；蚪M文庫(kù)技術(shù)是一種能夠?qū)蚪M進(jìn)行純化、貯存并在此基礎(chǔ)上進(jìn)行廣泛深入研究的分子生物學(xué)技術(shù),其中,細(xì)菌人工染色體(BAC)是20世紀(jì)90年代發(fā)展起來(lái)的新型DNA文庫(kù)載體技術(shù),具有穩(wěn)定性好,不易發(fā)生缺失重排,少有載體中毒效應(yīng)等優(yōu)點(diǎn),在染色體的組織進(jìn)化基因的組織結(jié)構(gòu)分析特定染色體基因的克隆基因組組織和基因表達(dá)基因組物理圖譜的構(gòu)建以及基因組測(cè)序等基因組研究的多個(gè)領(lǐng)域,BAC文庫(kù)都獲得了廣泛的應(yīng)用。 方法通過(guò)提取、純化得到鼠疫菌基因組DNA,經(jīng)限制性?xún)?nèi)切酶部分酶切后,與經(jīng)過(guò)末端去磷酸化處理的CopyControlTMpCC1BACTM Clone-ready載體按照一定摩爾比例相互連接,轉(zhuǎn)化TransforMax EPI300并涂板,經(jīng)藍(lán)白斑篩選,篩選陽(yáng)性克隆。通過(guò)分析陽(yáng)性克隆的末端測(cè)序結(jié)果,確認(rèn)克隆中插入片段中的來(lái)源。分別提取不同傳代的BAC克隆的質(zhì)粒DNA,用Hind III酶切分析比較指紋圖譜的變化,分析BAC克隆在繼代培養(yǎng)中是否穩(wěn)定。從所建庫(kù)中隨機(jī)挑選克隆,小量提取質(zhì)粒后脈沖電泳鑒定,以計(jì)算插入片段的平均長(zhǎng)度,空載率和覆蓋率。 結(jié)果末端測(cè)序及指紋圖譜分析結(jié)果,證明所建BAC文庫(kù)中插入片段確來(lái)源于宿主菌,且穩(wěn)定性良好。通過(guò)脈沖電泳及相關(guān)試驗(yàn)結(jié)果計(jì)算,該庫(kù)共包含3248個(gè)已測(cè)序克隆,平均插入片段為105 kb空載率為2%總的庫(kù)容為35Mb,約8倍于鼠疫菌基因組覆蓋率為97%。鼠疫強(qiáng)毒株BAC文庫(kù)構(gòu)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.

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R378

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本文編號(hào)：1383228