本文選題：淋病奈瑟菌 + CRISPR簇�。� 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：淋病奈瑟菌(Neisseria gonorrhoiae)俗稱淋球菌(gonococcus),是淋病的病原菌,主要通過(guò)性傳播,只感染人類,在男性常引起急性尿道炎,女性則一般表現(xiàn)為無(wú)癥狀感染,但病菌可向深部組織播散導(dǎo)致宮頸炎或不孕等嚴(yán)重后果。目前淋病沒(méi)有可用疫苗進(jìn)行預(yù)防,而治療又因淋病奈瑟菌耐藥性的日趨嚴(yán)重而越來(lái)越困難。全面理解淋病奈瑟菌的生物學(xué)特性可為淋病防治帶來(lái)新的靶點(diǎn)。成簇規(guī)律間隔短回文重復(fù)序列(Clustered Regularly Interspaced Short Palindromic Repeat,CRISPR)是近年發(fā)現(xiàn)的細(xì)菌獲得性免疫系統(tǒng),可使細(xì)菌防御外源DNA的再次入侵,為細(xì)菌抵抗不利環(huán)境因素提供了保障。該系統(tǒng)由CRISPR簇、Leader序列和Cas蛋白編碼基因等3部分組成,其中CRISPR簇由交替排列的保守性重復(fù)序列和特異性間隔序列組成。約90%古細(xì)菌和40%真細(xì)菌基因組中發(fā)現(xiàn)有CRISPR系統(tǒng),但淋病奈瑟菌的CRISPR系統(tǒng)尚未見(jiàn)報(bào)道。我們前期用生物信息學(xué)技術(shù)分析顯示在淋病奈瑟菌WHO-A株中存在CRISPR簇和Cas蛋白,并且發(fā)現(xiàn)CRISPR簇的間隔序列部分靶向自身基因,可能對(duì)細(xì)菌的生長(zhǎng)活動(dòng)具有某種調(diào)控作用。本研究旨在研究WHO-A株CRISPR簇與自身靶基因間的相互作用,為研究淋病奈瑟CRISPR系統(tǒng)的結(jié)構(gòu)與功能積累資料。一、淋病奈瑟菌WHO-A株CRISPR簇自身靶基因的篩選與克隆運(yùn)用CRISPRs Finder在線系統(tǒng)從前期測(cè)序獲得的WHO-A株基因組DNA序列中篩查CRISPR系統(tǒng),獲得P1～P6和CL等7個(gè)CRISPR簇,同時(shí)用重復(fù)區(qū)比對(duì)法進(jìn)行人工分析獲得1個(gè)CRISPR簇FAl。對(duì)這8個(gè)CRISPR簇的所有間隔序列進(jìn)行BLAST分析,發(fā)現(xiàn)這些間隔序列與多個(gè)淋病奈瑟菌自身基因具有同源性。進(jìn)一步按照間隔序列-靶基因間存在"g18 bp匹配序列的原則在已公布基因組序列的所有淋病奈瑟菌株中查找靶基因,再將所篩選的候選靶基因與WHO-A株基因組信息進(jìn)行同源性比較,從WHO-A株自身基因組中初篩出自身間隔序列所靶向的基因。考慮到靶基因中上游受到干擾時(shí)對(duì)其表達(dá)的影響更顯著,從上述靶基因中按照匹配位點(diǎn)位于中上游且至少有連續(xù)18 bp以上序列完全匹配的原則作進(jìn)一步篩查,最終獲得3個(gè)自身靶基因,即前噬菌體蛋白基因NGFG_01062、假定蛋白基因NGK_0072和菌毛相關(guān)蛋白基因NGK_2578。多個(gè)CRISPR簇中存在靶向NGK_2578基因的間隔序列,且CRISPR簇CL中存在多個(gè)間隔序列前后靶向該基因,提示淋病奈瑟菌CRISPR系統(tǒng)對(duì)菌毛可能發(fā)揮著重要的調(diào)控作用。運(yùn)用PCR技術(shù)分別擴(kuò)增了 3個(gè)自身靶基因,插入到pET-GFP質(zhì)粒中,重組載體pET-Targets-GFP導(dǎo)入到大腸埃希菌BL21中誘導(dǎo)表達(dá),通過(guò)SDS-PAGE和熒光顯微鏡檢測(cè)到靶蛋白-GFP的融合表達(dá)。二、利用重組大腸埃希菌分析淋病奈瑟菌CRISPR簇對(duì)自身靶基因的作用淋病奈瑟菌WHO-A株CRISPR系統(tǒng)的Leader與Cas蛋白的作用機(jī)制也不明確。但目前CRISPR/Cas9系統(tǒng)的結(jié)構(gòu)與功能研究非常深入,Addgene提供的成熟載體pCas9中Leader與Cas9的作用方式非常明確,可作為研究未知CRISPR簇功能的理想工具。為在大腸埃希菌中借助pCas9系統(tǒng)研究淋病奈瑟菌WHO-A株CRISPR簇與靶基因間的相互作用,我們合成了篩選的CRISPR簇間隔序列并插入到pCas9中的克隆位點(diǎn),構(gòu)建重組質(zhì)粒pCas9-S,導(dǎo)入含有靶基因表達(dá)載體pET-Targets-GFP的大腸埃希菌。重組菌用LB培養(yǎng)液傳代,每代細(xì)菌分別在氯霉素和卡那霉素LB平板上活菌計(jì)數(shù)以分析間隔序列是否引導(dǎo)Cas9對(duì)靶基因發(fā)揮核酸酶的切割作用。結(jié)果表明,在pCas9-S導(dǎo)入的最初并沒(méi)有發(fā)現(xiàn)其對(duì)靶基因產(chǎn)生明顯作用。隨著傳代的進(jìn)行,間隔序列P1S對(duì)靶基因NGFG_01062、CLS4對(duì)靶基因NGK_0072、CLS4和CLS5對(duì)NGK_578基因都表現(xiàn)出了抑制效果,受體菌在含有卡那霉素平板上的CFU明顯減少;qRT-PCR和SDS-PAGE分析顯示靶基因的轉(zhuǎn)錄和蛋白表達(dá)水平明顯下降。質(zhì)粒提取提示,間隔序列對(duì)靶基因產(chǎn)生作用的過(guò)程中可能造成了含靶基因重組質(zhì)粒的降解。間隔序列FAS1盡管不能降解靶基因NGK_2578但可抑制靶基因的轉(zhuǎn)錄從而降低目的蛋白的表達(dá)量。三、淋病奈瑟菌WHO-A株CRISPR簇的轉(zhuǎn)錄分析及基因敲除載體的構(gòu)建提取淋病奈瑟菌WHO-A株總RNA進(jìn)行RT-PCR檢測(cè)發(fā)現(xiàn),以針對(duì)CRISPR簇CL的特異性引物可以擴(kuò)增出明顯條帶;對(duì)總RNA進(jìn)行轉(zhuǎn)錄組測(cè)序,發(fā)現(xiàn)存在與CRISPR簇CL部分序列完全匹配的轉(zhuǎn)錄產(chǎn)物,提示CRISPR簇CL可轉(zhuǎn)錄形成前體crRNA。為構(gòu)建CRISPR簇CL基因敲除的淋病奈瑟菌突變株,設(shè)計(jì)了 6組針對(duì)CRISPR簇CL的sgRNA,插入pCas9的克隆位點(diǎn)獲得打靶載體pCas9-sgRNA;同時(shí)克隆了淋病奈瑟菌CRISPR簇CL基因片段作為待敲除的靶基因。大腸埃希菌中證明特異性pCas9-sgRNA降解CL基因,pCas9-sgRNA轉(zhuǎn)化淋病奈瑟菌WHO-A株構(gòu)建突變株的實(shí)驗(yàn)正在進(jìn)行中。另外還計(jì)劃應(yīng)用基于自殺載體的基因編輯技術(shù)對(duì)CRISPR簇CL進(jìn)行突變,利用卡那霉素抗性基因kan替換了 CL基因的中間部分,獲得的攜帶kan抗性篩選標(biāo)記的CL上下游同源臂插入自殺載體pGMB152,構(gòu)建了重組載體pGMB152△CL::Kan,為后期突變WHO-A株中CRISPR簇CL奠定了基礎(chǔ)。
[Abstract]:Neisseria gonorrhoeae (Neisseria gonorrhoiae), commonly known as gonococcus (gonococcus), is the pathogen of gonorrhea, mainly through sexual transmission, only infection of human, in men often cause acute urethritis, women generally appear to be asymptomatic infection, but the pathogen can spread to the deep tissue and cause severe consequences such as cervicitis or infertility. The gonorrhea is not available at present. A comprehensive understanding of the biological characteristics of Neisseria gonorrhoeae is a new target for the prevention and treatment of Neisseria gonorrhoeae. Clustered Regularly Interspaced Short Palindromic Repeat, CRISPR) is the finer in recent years. The bacterial acquired immune system can enable the bacteria to re invade the exogenous DNA and provide a guarantee for the bacterial resistance to adverse environmental factors. The system consists of 3 parts, such as CRISPR cluster, Leader sequence and Cas protein encoding gene, in which the CRISPR cluster is composed of alternate sequence of conserved repeat sequences and specific interval sequences. About 90% palaeobacteria and 40% The CRISPR system was found in the eubacterial genome, but the CRISPR system of Neisseria gonorrhoeae has not yet been reported. In the earlier period of our bioinformatics analysis, we showed that there were CRISPR and Cas proteins in Neisseria gonorrhoeae WHO-A strain, and that the interval sequence of the CRISPR cluster was targeted to the self gene, which might have a certain effect on the growth of bacteria. The purpose of this study is to study the interaction between the CRISPR cluster and the target gene of the WHO-A strain, and to study the structure and function accumulation of the Neisseria gonorrhoeae CRISPR system. 1, the screening and cloning of the CRISPR cluster of Neisseria gonorrhoeae WHO-A strain CRISPR cluster, the genomic DN of WHO-A strain obtained from the early sequencing of the CRISPRs Finder system In the A sequence, the CRISPR system was screened, 7 CRISPR clusters, such as P1 to P6 and CL, were obtained. At the same time, 1 CRISPR cluster FAl. were used to analyze all the interval sequences of the 8 CRISPR clusters by artificial analysis of the repeat area alignment method. It was found that these interval sequences were homologous to the self base of Neisseria gonorrhoeae, and further according to the interval sequence - target. The principle of "G18 BP matching sequence" is found in all the gonorrhoeae strains that have been published in the genome sequence, and then the target genes are compared with the genomic information of the WHO-A strain, and the target genes are initially screened out from the WHO-A genome of the WHO-A strain. The upstream genes are taken into account in the upstream of the target gene. The effect of interference on its expression is more significant. Further screening is made from the target gene in the upper and middle reaches of the target gene in the middle and upper reaches of the middle and at least 18 bp consecutive sequences. The final 3 target genes, the pre phage gene NGFG_01062, the hypothetical protein gene NGK_0072 and the pili related protein gene N, are finally obtained. The interval sequence of the target NGK_2578 gene exists in multiple CRISPR clusters of GK_2578., and the target gene is targeted before and after multiple interval sequences in the CRISPR cluster CL, suggesting that the CRISPR system of Neisseria gonorrhoeae may play an important regulatory role in the pilus. 3 target genes were amplified by PCR technique and inserted into the pET-GFP plasmid, and the recombinant vector was inserted. PET-Targets-GFP was introduced into the Escherichia coli BL21 to induce expression, and the fusion expression of target protein -GFP was detected by SDS-PAGE and fluorescence microscope. Two, the effect mechanism of Leader and Cas protein of Neisseria gonorrhoeae WHO-A strain CRISPR system of Neisseria gonorrhoeae WHO-A strain of Neisseria gonorrhoeae was analyzed by recombinant Escherichia coli. But at present, the structure and function of the CRISPR/Cas9 system are deeply studied. The mode of action of Leader and Cas9 is very clear in the mature carrier pCas9 provided by Addgene. It can be used as an ideal tool to study the function of unknown CRISPR cluster. The interaction between CRISPR cluster and target gene of Neisseria gonorrhoeae WHO-A strain of Neisseria gonorrhoeae in Escherichia coli is studied by pCas9 system. We synthesized the selected CRISPR cluster interval sequences and inserted the clones in the pCas9, constructed the recombinant plasmid pCas9-S, and introduced the Escherichia coli containing the target gene expression vector pET-Targets-GFP. The recombinant bacteria were passed through the LB culture medium, and each generation of bacteria counted on the chloramphenicol and kanamycin LB plate respectively to analyze the interval sequence. Whether the target gene plays the nuclease of the target gene was guided by Cas9. The results showed that the target gene had no obvious effect on the target gene at the beginning of the pCas9-S introduction. With the passage of the passage, the interval sequence P1S showed the target gene NGFG_01062, CLS4 to the target gene NGK_0072, CLS4 and CLS5 to the NGK_578 gene and the receptor bacteria. QRT-PCR and SDS-PAGE analysis showed that the transcription and protein expression level of the target gene decreased obviously on the kanamycin tablet containing the kanamycin plate. The plasmid extraction could lead to the degradation of the recombinant plasmid containing the target gene in the process of the effect of the interval sequence on the target gene. The septum sequence FAS1 could not degrade the target gene NGK_2578. But it could inhibit the transcription of the target gene and reduce the expression of the target protein. Three, the transcriptional analysis of the CRISPR cluster of Neisseria gonorrhoeae WHO-A strain and the construction of the gene knockout vector to extract the total RNA of Neisseria gonorrhoeae WHO-A strain were detected by RT-PCR detection. The specific primers for the CRISPR cluster CL could be amplified by the specific primers, and the total RNA was transcribed. Sequencing, it was found that there was a transcriptional product that was completely matched with the partial sequence of the CRISPR cluster CL, suggesting that the CRISPR cluster CL can be transcribed crRNA. as a CRISPR cluster CL gene knockout mutant of Neisseria gonorrhoeae, and 6 groups of sgRNA for CRISPR cluster CL were designed and the target carrier pCas9-sgRNA was inserted into the pCas9 clone site, and gonococcal Nai was cloned at the same time. The CL gene fragment of CRISPR cluster was used as the target gene to be knocked out. Escherichia coli showed that specific pCas9-sgRNA degrade CL gene and pCas9-sgRNA transformation of Neisseria gonorrhoeae WHO-A strain was in progress. In addition, the gene editing technique based on suicide vector was also planned to mutate CRISPR cluster CL and use kanamycin. The vegetal resistance gene Kan replaced the middle part of the CL gene, and the CL upstream and downstream homologous arm pGMB152 was inserted with the Kan resistance screening marker, and the recombinant vector pGMB152 Delta CL:: Kan was constructed, which laid the foundation for the CRISPR cluster CL in the later mutant WHO-A strain.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R378
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本文編號(hào)：1805338