淋病奈瑟菌CRISPR簇与自身靶基因间相互作用的初步研究

发布时间：2018-04-26 08:59

  本文选题：淋病奈瑟菌 + CRISPR簇　； 参考：《扬州大学》2017年硕士论文

【摘要】：淋病奈瑟菌(Neisseria gonorrhoiae)俗称淋球菌(gonococcus),是淋病的病原菌,主要通过性传播,只感染人类,在男性常引起急性尿道炎,女性则一般表现为无症状感染,但病菌可向深部组织播散导致宫颈炎或不孕等严重后果。目前淋病没有可用疫苗进行预防,而治疗又因淋病奈瑟菌耐药性的日趋严重而越来越困难。全面理解淋病奈瑟菌的生物学特性可为淋病防治带来新的靶点。成簇规律间隔短回文重复序列(Clustered Regularly Interspaced Short Palindromic Repeat,CRISPR)是近年发现的细菌获得性免疫系统,可使细菌防御外源DNA的再次入侵,为细菌抵抗不利环境因素提供了保障。该系统由CRISPR簇、Leader序列和Cas蛋白编码基因等3部分组成,其中CRISPR簇由交替排列的保守性重复序列和特异性间隔序列组成。约90%古细菌和40%真细菌基因组中发现有CRISPR系统,但淋病奈瑟菌的CRISPR系统尚未见报道。我们前期用生物信息学技术分析显示在淋病奈瑟菌WHO-A株中存在CRISPR簇和Cas蛋白,并且发现CRISPR簇的间隔序列部分靶向自身基因,可能对细菌的生长活动具有某种调控作用。本研究旨在研究WHO-A株CRISPR簇与自身靶基因间的相互作用,为研究淋病奈瑟CRISPR系统的结构与功能积累资料。一、淋病奈瑟菌WHO-A株CRISPR簇自身靶基因的筛选与克隆运用CRISPRs Finder在线系统从前期测序获得的WHO-A株基因组DNA序列中筛查CRISPR系统,获得P1～P6和CL等7个CRISPR簇,同时用重复区比对法进行人工分析获得1个CRISPR簇FAl。对这8个CRISPR簇的所有间隔序列进行BLAST分析,发现这些间隔序列与多个淋病奈瑟菌自身基因具有同源性。进一步按照间隔序列-靶基因间存在"g18 bp匹配序列的原则在已公布基因组序列的所有淋病奈瑟菌株中查找靶基因,再将所筛选的候选靶基因与WHO-A株基因组信息进行同源性比较,从WHO-A株自身基因组中初筛出自身间隔序列所靶向的基因。考虑到靶基因中上游受到干扰时对其表达的影响更显著,从上述靶基因中按照匹配位点位于中上游且至少有连续18 bp以上序列完全匹配的原则作进一步筛查,最终获得3个自身靶基因,即前噬菌体蛋白基因NGFG_01062、假定蛋白基因NGK_0072和菌毛相关蛋白基因NGK_2578。多个CRISPR簇中存在靶向NGK_2578基因的间隔序列,且CRISPR簇CL中存在多个间隔序列前后靶向该基因,提示淋病奈瑟菌CRISPR系统对菌毛可能发挥着重要的调控作用。运用PCR技术分别扩增了 3个自身靶基因,插入到pET-GFP质粒中,重组载体pET-Targets-GFP导入到大肠埃希菌BL21中诱导表达,通过SDS-PAGE和荧光显微镜检测到靶蛋白-GFP的融合表达。二、利用重组大肠埃希菌分析淋病奈瑟菌CRISPR簇对自身靶基因的作用淋病奈瑟菌WHO-A株CRISPR系统的Leader与Cas蛋白的作用机制也不明确。但目前CRISPR/Cas9系统的结构与功能研究非常深入,Addgene提供的成熟载体pCas9中Leader与Cas9的作用方式非常明确,可作为研究未知CRISPR簇功能的理想工具。为在大肠埃希菌中借助pCas9系统研究淋病奈瑟菌WHO-A株CRISPR簇与靶基因间的相互作用,我们合成了筛选的CRISPR簇间隔序列并插入到pCas9中的克隆位点,构建重组质粒pCas9-S,导入含有靶基因表达载体pET-Targets-GFP的大肠埃希菌。重组菌用LB培养液传代,每代细菌分别在氯霉素和卡那霉素LB平板上活菌计数以分析间隔序列是否引导Cas9对靶基因发挥核酸酶的切割作用。结果表明,在pCas9-S导入的最初并没有发现其对靶基因产生明显作用。随着传代的进行,间隔序列P1S对靶基因NGFG_01062、CLS4对靶基因NGK_0072、CLS4和CLS5对NGK_578基因都表现出了抑制效果,受体菌在含有卡那霉素平板上的CFU明显减少;qRT-PCR和SDS-PAGE分析显示靶基因的转录和蛋白表达水平明显下降。质粒提取提示,间隔序列对靶基因产生作用的过程中可能造成了含靶基因重组质粒的降解。间隔序列FAS1尽管不能降解靶基因NGK_2578但可抑制靶基因的转录从而降低目的蛋白的表达量。三、淋病奈瑟菌WHO-A株CRISPR簇的转录分析及基因敲除载体的构建提取淋病奈瑟菌WHO-A株总RNA进行RT-PCR检测发现,以针对CRISPR簇CL的特异性引物可以扩增出明显条带;对总RNA进行转录组测序,发现存在与CRISPR簇CL部分序列完全匹配的转录产物,提示CRISPR簇CL可转录形成前体crRNA。为构建CRISPR簇CL基因敲除的淋病奈瑟菌突变株,设计了 6组针对CRISPR簇CL的sgRNA,插入pCas9的克隆位点获得打靶载体pCas9-sgRNA;同时克隆了淋病奈瑟菌CRISPR簇CL基因片段作为待敲除的靶基因。大肠埃希菌中证明特异性pCas9-sgRNA降解CL基因,pCas9-sgRNA转化淋病奈瑟菌WHO-A株构建突变株的实验正在进行中。另外还计划应用基于自杀载体的基因编辑技术对CRISPR簇CL进行突变,利用卡那霉素抗性基因kan替换了 CL基因的中间部分,获得的携带kan抗性筛选标记的CL上下游同源臂插入自杀载体pGMB152,构建了重组载体pGMB152△CL::Kan,为后期突变WHO-A株中CRISPR簇CL奠定了基础。
[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.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378
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本文编号：1805338