【作者】 孙卫忠；

【导师】 胡福泉；

【作者基本信息】 第三军医大学 ， 微生物， 2007， 博士

【摘要】 研究背景与意义:二十世纪九十年代人类基因组计划(human genome project,HGP)的提出及完成,极大地促进和带动了一系列生物基因组计划的蓬勃发展。截止到目前为止,GenBank已经登录了数千种生物的基因组序列,并且还有数百种生物的基因组测序正在进行之中。在登录的基因组数据中,有的完成了初步注释,有的仅仅是完成了测序工作。尽管测序工作进展神速,但基因功能研究明显滞后,这给科研工作者提出了新的课题。因此,基因功能研究成为了后基因组学或功能基因组学时代的艰巨任务之一。本室在前期工作中分离鉴定了3株铜绿假单胞菌噬菌体,分别命名为PaP1、PaP2和PaP3(pseudomonas aeruginosa phage)。目前,溶源性噬菌体PaP2和PaP3全基因组测序及基因初步注释工作已经完成,并提交到GenBank。裂解性噬菌体PaP1基因组也已经完成了部分测序。根据分析,这三种噬菌体所预测的基因中大多数为功能未知基因。因而,展开对这些基因的分析及功能验证成为我们面临的重要任务。在功能基因研究中,我们首先把目标集中在与噬菌体裂解细菌有关的基因上。这是因为,噬菌体是以细菌为宿主的病毒,噬菌体在感染宿主菌时,必须穿过细菌的细胞壁肽聚糖层,或者将其核酸注入到宿主菌体内进行复制。噬菌体在完成复制、组装后,又必须破坏宿主肽聚糖层,释放子代噬菌体,进而感染新的细菌。因此,噬菌体完成其繁殖,必须“一进一出”两次穿越宿主菌的细胞壁屏障。与单链(DNA/RNA)噬菌体不同的是,双链DNA噬菌体感染宿主的结果,往往最终杀死宿主菌。因此,噬菌体在感染过程中必然产生一种能够破坏宿主细胞壁肽聚糖层的相关酶类。而细胞壁是细菌赖以生存的基础,能够破坏细胞壁的酶类也就有可能成为潜在的抗感染药物。根据文献报道,控制双链DNA噬菌体进出宿主细胞的物质是一种由噬菌体编码的蛋白,称作内溶素或者肽聚糖解聚酶(Endolysin或者Lysin,简写为elys)。内溶素作为一种酶蛋白,可以破坏噬菌体宿主菌的细胞壁肽聚糖。因而,内溶素有可能成为某些致病细菌特别是耐药性致病菌的潜在的抗感染药物。另外,大部分噬菌体的内溶素由于缺少信号肽不能够穿透宿主细胞膜,通常需要噬菌体编码的一种穴蛋白(Holin)辅助才能够穿过宿主细胞膜,进入周间质进而作用于宿主细胞壁肽聚糖层。因此,对于噬菌体内溶素及穴蛋白进行广泛深入的研究不但有助于了解噬菌体与宿主的相互作用、揭示生命的多样性,而且将内溶素作为生物制剂,应用于噬菌体治疗(phage therapy)也有重要的研究意义。材料与方法:收集GenBank中已经登录的全部铜绿假单胞菌双链DNA噬菌体基因组序列(含我室提交的噬菌体PaP2和PaP3基因组序列)及我室未提交的噬菌体PaP1部分基因组序列。首先,初步分析各基因组组成特点,并进行比较基因组分析;其次,通过对所有基因组可能编码的ORF进行BLAST检索,从而推定所有可能的内溶素及穴蛋白基因;再次,对发现的铜绿假单胞菌噬菌体内溶素及穴蛋白进行比较、归类、三级结构预测、系统发生进化分析及跨膜结构域预测;最后,克隆、表达推定的铜绿假单胞菌噬菌体PaP1内溶素基因,纯化基因表达产物,分析这些表达产物对底物肽聚糖的降解活性及抑菌作用,通过实验证实推定的内溶素的生物学功能。结果与讨论:1.铜绿假单胞菌噬菌体基因组比较:经过搜集整理,共有20种铜绿假单胞菌双链DNA噬菌体基因组序列。除10种铜绿假单胞菌噬菌体基因组已完成了初步注释外,其余铜绿假单胞菌噬菌体基因组仅仅是完成了测序工作,未作任何注释。通过对基因组序列比较分析,除这三对噬菌体外,其它铜绿假单胞菌噬菌体基因组序列相互之间相似性极低。这三对噬菌体分别是PA7与PA16、119X与PaP2、SD1-M与phiKZ,其中噬菌体PA7与PA16基因组序列之间相似性达99%以上,噬菌体119X和PaP2基因组序列之间相似性为93%,噬菌体SD1-M与phiKZ基因组序列之间相似性为99%。这些相似性的序列有的表现为大片段完全相同,呈嵌合体状态;有的表现为突变碱基呈点状分散,且基因组两端序列变化显著。这种结构模式表明,噬菌体基因组的进化与其它物种的进化类似,分为渐变式和爆发式。其中爆发式进化可以用模块进化理论(modular evolution)来解释:基因或蛋白的进化并不是通过连续的一个个的核苷酸或者氨基酸的替换,而是通过装配已经存在的编码蛋白多肽的核苷酸区段完成的,而进化的结果则表现在噬菌体基因组呈镶嵌型结构,且基因组两端序列变化显著。2.铜绿假单胞菌噬菌体内溶素基因的推定:根据铜绿假单胞菌噬菌体在Genbank登录的序列及基因注释,已有12种内溶素基因被发现与命名。而我们对20种铜绿假单胞菌噬菌体基因组重新分析,新发现8种内溶素基因。这8种噬菌体内溶素基因分别是噬菌体F10的ORF37273～37890、噬菌体的F8 ORF24127～26709及ORF34978～35637、噬菌体M6的ORF13111～13932、噬菌体Pa11的ORF21764～22279、噬菌体SD1-M的ORF147382~148161及ORF190416~197138、噬菌体PaP1的ORF25043～25603。3铜绿假单胞菌噬菌体内溶素分类:对20种噬菌体内溶素基因编码的氨基酸序列经过clustalw多重序列比对后生成的进化树结果来看,铜绿假单胞菌噬菌体内溶素可以分为4类:包括N-乙酰胞壁酸酶类、葡萄糖苷酶类、转糖基酶类和未知功能类。新发现的8种噬菌体内溶素中,属于N-乙酰胞壁酸酶类的内溶素只有1种,是噬菌体pa11的ORF21764～22279编码产物;属于葡萄糖苷酶类的内溶素有2种,分别是噬菌体F8的ORF34978～35637和F10的ORF37273～37890编码的产物;属于转糖基酶类的内溶素有4种,分别是噬菌体M6的ORF13111～13932、噬菌体F8的ORF24127~26706及噬菌体SD1-M的ORF147382~148161及ORF190416~197138编码的产物均;而噬菌体PaP1的ORF25043～25603编码的产物由于同时与肽链内切酶和酰氨酶在N-端序列上有一定的相似性,目前尚不能归类,本文将之归为未知功能类。4.铜绿假单胞菌噬菌体穴蛋白基因推定:在20种噬菌体中,已有3种噬菌体编码穴蛋白的基因被注释。我们对20种噬菌体基因组重新分析,新发现了2种穴蛋白基因,分别是噬菌体F10的ORF36944~37276及噬菌体M6的ORF12887~13219。5种穴蛋白的基因序列及编码的氨基酸序列具有共同的基本特征:编码穴蛋白的基因位于内溶素基因的上游;通常含有至少两个疏水区和一段较短的连接序列;蛋白质多肽链氨基酸残基个数在71～111之间。此外穴蛋白基因在噬菌体基因组内与内溶素基因均有多个碱基的重叠现象(genes overlap)。在其它未发现穴蛋白噬菌体的基因组序列中,均能发现一个或几个编码含有跨膜区结构的基因,这些含有跨膜区结构的基因是否是噬菌体穴蛋白的候选基因值得深入研究。5.噬菌体PaP1内溶素基因—ely的克隆、表达与纯化:本文选择铜绿假单胞菌噬菌体PaP1内溶素基因进行验证。通过对PCR反应体系和条件的优化,以噬菌体PaP1基因组DNA为模板,通过PCR扩增获得噬菌体PaP1内溶素基因全长序列。将该基因构建到表达载体pQE31中,然后转化大肠埃希菌M15(pREP4)并诱导表达。在一定范围内随IPTG浓度的增加,重组蛋白表达率增加,至0.4～0.6mM时基本达到最高峰值,重组蛋白表达最佳诱导时间为6h,重组蛋白表达形式主要以可溶性状态存在。经过Ni-NTA亲和柱层析、UNOsphere-S阳离子交换层析及分子筛脱盐后,获得了与预测分子量大小相符的单一条带的目标蛋白。6.底物的提取与ELY-6H重组融合蛋白酶活性检测:①底物提取:采用SDS(4%,w/v)煮沸方法提取铜绿假单胞菌细胞壁肽聚糖成分。②酶活性检测方法:采用酶谱电泳法、扩散法及比浊度法检测ELY-6H重组融合蛋白对细胞壁肽聚糖层的降解活性。③结果:酶谱电泳方法结果显示ELY-6H重组融合蛋白及阳性对照鸡溶菌酶标准品对铜绿假单胞菌细胞壁肽聚糖均有降解作用;扩散法及比浊度法结果显示ELY-6H重组融合蛋白对底物肽聚糖的降解活性优于鸡溶菌酶活性。同鸡溶菌酶相比,ELY-6H重组蛋白能够将周围底物部分彻底降解,形成的透明圈较亮且边缘规则,鸡溶菌酶处理后形成的透明圈直径稍大,但边缘呈弥散状,对肽聚糖降解不完全,且经ELY-6H重组蛋白处理的底物溶液吸光度值变化范围大于鸡溶菌酶处理结果。7.ELY-6H重组融合蛋白的抑菌活性检测:将ELY-6H重组融合蛋白分别处理培养的铜绿假单胞菌、大肠埃希菌、金黄色葡萄球菌(ATCC 25923)和溶壁微球菌等活菌。结果显示,ELY-6H重组融合蛋白除了对金黄色葡萄球菌有一定的抑菌效果外,对其它革兰阳性细菌及革兰阴性细菌没有抑菌活性。结论本研究通过对铜绿假单胞菌噬菌体内溶素的基因推定,共发现20种内溶素基因和6种穴蛋白基因,其中8种内溶素基因和2种穴蛋白基因为本研究新发现基因。推定的内溶素基因分属于4个类群:转糖基酶类、N-乙酰胞壁酸酶类、N-乙酰葡萄糖苷酶类和功能未知类。选择一推定的噬菌体PaP1 ely基因进行克隆和表达,同时对表达的ELY-6H重组融合蛋白进行了纯化,得到较纯的重组融合蛋白。对该重组融合蛋白对底物肽聚糖的降解活性检测及抑菌活性检测。结果表明,推定的噬菌体PaP1重组内溶素融合蛋白对铜绿假单胞菌细胞壁肽聚糖具有降解活性,并且对金黄色葡萄球菌活菌具有一定的抑菌活性。该研究从实验方面证实了推定的噬菌体PaP1内溶素基因的生物学功能。但该基因的工程菌表达产物在医学抗感染方面可能不具有直接应用意义。更多还原

【Abstract】 Research background and objectives:The human genome project (HGP) has greatly promoted the development of thousands of organism genome projects since the year of 1990. Up to now, there are thousands of organism genomes have been submitted to GenBank, and there still hundreds of genome sequencing projects were under way. However, the functional genomics research has lagged far behind the genome sequences research and there only few genes were functionally verified by experiment. In addition, there are still lots of organism genomes sequences were totally not annotated, and needed to be verified. The main interests of the biologists have been transferred from the structural genomics to the functional genomics which focus on elucidating the function of genes.Previously three new bacteriophages (phages) of pseudomonas aeruginosa were isolated from hospital sewage in our laboratory and named as PaP1, PaP2 and PaP3 (PaP: pseudomonas aeruginosa phage) respectively. Then the whole genome of the temperate phage PaP2 and PaP3 were sequenced and submitted to GenBank. The partial genome of PaP1, a virulent phage, was also sequenced and kept in our lab. Since most predicated ORFs (open reading frames) of the three phages are functionally unknown, it is necessary for us to analyze and identify these unknown genes of the three phages.During their infection from outside or offspring release from inside of the host cells, phages must elaborate enzymes to destroy the cell wall of their bacterial hosts. The bacterial cell wall is of great significance to the survival of bacteria, so if a kind of enzyme which has peptidoglycan lytic activity, was found it would be potentially used as a useful antibacterial agent in the future. In this research, we firstly and foremostly focused our work on the bacteriophage enzymes which can cause the lysis of the host cell wall. To the best of our knowledge, phages are the bacteria viruses which elaborate Endolysins to degrade the cell wall of their host. Endolysins (or lysins) are double-stranded DNA bacteriophage encoded peptidoglycan enzymes and they were produced in phage-infected bacterial cells at the end of the life cycle. Endolysins are also capable of degrading peptidoglycan when applied to the bacterial cell wall, and cause a rapid lysis of the bacterial cell. Because of their unique ability to cleave peptidoglycan in a generally species-specific manner, Endolysins represent a novel class of antibacterial agents. In general, Endolysins have no secretary signal sequence. They were unable to across the cytoplasmic membrane directly and thus accumulate in the cytoplasm, so another protein which was named as Holin is needed for the release of Endolysin. For the reason that bacteriophage Endolysins can be potentially used as the antibacterial agents, a deep insight into their enzymic function is of much helpful not only for investigating the interaction between phages and their host, but also for revealing the diversity of phages, and exploiting their antibacterial application for phage therapy in the future.Materials and Methods:Firstly, the P. aeruginosa bacteriophage genome sequences, which include all the ds-DNA phages that had been submitted to GenBank and all the genomes that had been sequenced by our lab, were collected. Secondly, the genome composition, sequences similarity of these phages was analyzed. In order to find all the endolysin and holin genes, open reading frame (ORF) predication, similarity alignments of the predicated gene sequences were then performed by bio-tools. Thirdly the predicted endolysin and holin genes were analyzed by the methods such as sequence similarity, phylogeny group classification tertiary structure predication and so on. Lastly, one predicated endolysin gene of bacteriophage PaP1 was cloned, expressed and purified. The purified recombinant protein was then verified subsequently.Results and discussion1. Analysis of the whole genome sequences of P. aeruginosa bacteriophages. There are total 20 P. aeruginosa ds-DNA bacteriophage genome sequences were collected. Of the 20 phages, genome sequences of 10 phages have been analyzed and annotated. The sequences of other 10 phages were unannotated till now. In this research, the genome composition, sequences similarity of the 20 phages was performed. Results reveal that with three exceptions, the P. aeruginosa phages show little sequence relatedness to each other. These three exceptions are PA7 and PA16 (more than 99% identity), 119X and PaP2 (93% identity), and SD1-M and phiKZ (more than 99% identity). We predicated that the evolution of these phages is evolved not only by gradual evolution, by which phages change their genomes by single or few base pairs gradually, but also by explosive evolution, by which phages change their genomes by large segments. The explosive evolution can also be explained by the theory of modular evolution, and the results of this evolution method make the genome of phages more chimeric like (or mosaic like), and there are more mutations of the terminal region than of the middle region in the phage genomes.2. Enzymatic function predication of the P. aeruginosa bacteriophage Endolysins. There are 12 endolysin genes had been reported or nominated in GenBank. Based on the ORF predication and homology alignments of the 20 phage genome sequences, 8 endolysin genes were newly found from 6 phages. These 8 endolysin genes are the ORF37273～37890 of bacteriophage F10, the ORF24127～26709 and ORF34978～35637F8 of bacteriophage F8, the ORF13111～13932 of bacteriophage M6, the ORF21764～22279 of bacteriophage Pa11, the ORF147382~148161 and ORF190416~197138 of bacteriophage SD1-M, the ORF25043～25603 of bacteriophage PaP1.3 classifications of the P. aeruginosa bacteriophage Endolysins: Results show that all the Endolysins of these phages can be divided into four categories which include muramidases, glucosaminidases, transglycosylases and unknown categories. Of the 8 newly found Endolysins, the ORF21764～22279 of bacteriophage pa11 is belong to muramidase; the ORF34978～35637 of phage F8 and the ORF37273～37890 of phage F10 are belong to glucosaminidase; the ORF13111～13932 of phage M6, the ORF24127~26706 of phage F8 and the two Endolysin (ORF147382~148161 and ORF190416~197138) of phage SD1-M are belong to transglycosylase; the ORF25043～25603 of phage PaP1 is belong to the unknown categories due to its strange structures and unknown enzymatic functions.4. Predication of holin genes of the P. aeruginosa bacteriophages. There are 3 holin genes had been reported or nominated in GenBank. Based on ORF predication and homology alignments of these phage genome sequences, 2 holin genes were newly found from 2 phages. The 2 newly found holins are the ORF36944~37276 of Bacteriophage F10 and the ORF12887~13219 of bacteriophage M6. All the Holin of P. aeruginosa bacteriophages have the common character of other bacteriophage Holin’s such as there are at least two putative transmembrane domains separated by short linker sequences; holin genes are usually located upstream of the endolysin genes of the phage genome; the encoded amino acid sequences length is between 71 and 111 and there are gene overlap by several base pairs between the holin gene and the endolysin gene. Although the holin genes from most other phages were not find in this research, but there are some genes which also have one or more transmembrane domains of those phage genomes and whether or not these function unknown genes are the holin gene candidates is needed to be further studied.5. Molecular cloning, expression and purification of the bacteriophage PaP1 ely gene: The whole PaP1 genome DNA was used as template to amplify the ely gene. The amplification products were finally constructed into the expression vector pQE31 and transformed into the competent cells of E. coli M15 (pREP4) and induced by IPTG. The recombinant ELY-6H can be expressed at high level after 6 hours treated with 0.5mmol/L IPTG, and most of the fusion protein was of a soluble form. The soluble fractions of the six-His-tagged fusion proteins was purified by nickel-affinity chromatography using gravity-flow, and then repurified on a cation exchange resin and desalted on a molecular-sieve chromatography.6. Enzymatic activity assay of the ELY-6H. The peptidoglycan substrate of P. aeruginosa purification was obtained by boiling P. aeruginosa cells in SDS (4%, w/v). Enzymatic activity was detected by zymography assay, gel diffusion assay and turbidimetry assay respectively. The results of enzymatic assay show that both ELY-6H and hen egg white lysozyme can cause the lysis of the purified P. aeruginosa peptidoglycan. The results of gel diffusion assay show that the digest zone of the ELY-6H to the peptidoglycan substrate is more clearly against the background than that of hen egg white lysozyme, and the results of turbidimentry assay show that the absorption of the peptidoglycan solutions which treated with ELY-6H changes greatly than that treated with hen egg white lysozyme.7.Antibacterial activity assay of the ELY-6H. The inhibition activity assay of ELY-6H was applied exogenously to the cultures of P. aeruginosa, S. aureaus (ATCC 25923), Micrococcus lysoleikticus and E. coli JM109, respectively. Results show that the bacterial lawn growth on the agar of Gram-positive bacteria S. aureaus (ATCC 25923) was inhibited by both ELY-6H and hen egg white lysozyme. The inhibition of hen egg white lysozyme to the growth of S. aureaus (ATCC 25923) was stronger than that of ELY-6H. The inhabitation of ELY-6H to the lawn growth of the other bacteria (such as P. aeruginosa and E. coli BL21) was unobvious.Conclusions:8 new endolysin genes and 2 holin genes were found by the analysis of the 20 P. aeruginosa bacteriophage genomes. All the Endolysins of these phages can be divided into four categories including transglycosylases, muramidases, glucosaminidases and the unknown categories. The PaP1 endolysin gene was cloned and expressed in E. coli M15 (pREP4). The fusion protein ELY-6H was purified and its enzymatic activity was detected. Results show that the cell wall of P. aeruginosa can be degraded by the purified recombinant fusion protein ELY-6H. The antibacterial activity to its host was not found of the purified recombinant protein in this research, but it can inhibit the growth of Staphylococcus aureaus. All the results of this research will bring light to the identification the bacteriophage Endolysin and make it to be used as the potential antibacterial materials in the future.更多还原