超强耐药粪肠球菌噬菌体裂解酶和柯萨奇病毒A组16型病毒样颗粒的表达、纯化和结晶学研究

发布时间：2018-06-16 16:10

本文选题：粪肠球菌 + 噬菌体裂解酶　；参考：《吉林大学》2017年硕士论文

【摘要】：粪肠球菌是人或动物肠道、口腔和生殖道的正常菌群,正常情况下,共生的粪肠球菌对宿主没有致病性。但当其异位寄生时可引起败血症、尿路感染、化脓性腹部感染和心内膜炎等疾病,随着粪肠球菌耐药性加重,其所引起的感染更加难以控制,迫切需要研究新型抗菌制剂。噬菌体的出现为我们提供了一个新的思路,对于噬菌体及噬菌体裂解酶的研究也显现出巨大的应用价值。科学家从各种感染了噬菌体的致病菌,包括肺炎链球菌、炭疽杆菌、乳酸杆菌、金黄葡萄球菌等都分离出噬菌体。但随着抗生素的出现,人们的注意力又转移到抗生素上面来,从而忽略了细菌的天敌噬菌体。随着病原细菌的超强、广谱耐药性问题使得噬菌体疗法用于耐药性细菌感染的治疗成为研究的热点。噬菌体及其裂解酶可以特异性的裂解细菌,不会侵染真核细胞,也不会破坏机体的正常菌群,是新概念的抗菌物质。对噬菌体及其裂解酶的深入研究,将会开发出新型抗菌制剂,用于细菌性疾病的治疗。前期实验中,我们由临床样本中分离得到了对耐万古霉素粪肠球菌具有高效裂解活性的新噬菌体,通过全基因组序列测定和分析,确定了噬菌体的裂解酶基因,通过基因工程表达获得了裂解酶LysN10。实验共选取36株粪肠球菌,LysN10可以裂解其中32株菌,且LysN10与LysGH的CHAP片段在氨基酸序列具有一定的同源性。裂解酶对粪肠球菌表现出广谱、高效的裂解活性,显示了该裂解酶治疗耐药性粪肠球菌感染的潜力。为通过晶体衍射方法解析噬菌体裂解酶LysN10的结构,本论文首先获得了pET15b-N10的原核表达载体,并于大肠杆菌中表达。通过表达条件的摸索、亲和层析、分子筛层析等方法得到了纯度达95%的目标蛋白。对于全长LysN10我们采用蛋白质结晶条件筛选试剂盒进行晶体结晶条件的筛选,通过连续一个月的观察,未能获得蛋白晶体。通过对裂解酶和同源蛋白结构的分析,我们推测该裂解酶由两个独立的结构域组成,结构域间通过一个较长的柔性linker连接。这样的结构特点,造成该全长蛋白很难结晶。为解决这一问题,我们借鉴同源蛋白结构解析方式,通过序列分析,分别从结合结构域和功能结构域对全长蛋白设计截短,构建了 3个不同位点的截断突变体,命名为NF418,NR438和NR492。对已经构建好的重组质粒pET15b-NF418,pET15b-NR438和pET15b-NR492分别进行表达、纯化以及结晶条件的筛选和优化。通过对296种结晶条件的筛选,我们最终获得突变体NF418的晶体。这为完全解析LysN10的结构奠定基础。手足口病(hand,foot and mouth disease,HFMD)是由肠道病毒引起的一种传染病,柯萨奇病毒A组16型(CoxsackievirusA16,CA16)是CV的主要成员之一,1951年首次在南非被成功分离。CA16所致的手足口病症状相对较轻,且与由EV71引起的手足口病的具体症状难以区别,另外,患儿和隐性感染者均为传染源,成人感染后成为隐性感染者和无症状带毒者,本身不发病,但可作为潜在的传染源将病毒传染给幼儿,这也是造成疾病传播难以控制的主要原因之一。许多病毒结构蛋白都具有自动组装成VLPs的能力,在形态结构上与天然的病毒颗粒相似,具有很强的免疫原性和生物学活性。由于VLPs不含有病毒遗传物质,因此不具有感染性,其中有些已经作为疫苗成功应用于临床。由于VLPs表面能够重复且高密度展示外源性表位从而引发强有力的免疫应答,因此它对于多种疾病来说都是一种可开发理想的疫苗形式。研究表明,CA16病毒是由60个VP1,VP2,VP3以及VP4衣壳蛋白构成的亚单位共同形成一个二十面体的球形颗粒。与其他小RNA病毒相同,其VP1,VP2,和VP3蛋白各自组成衣壳的一个亚单位,此亚单位又构成一个五聚体,12个五聚体最终构成衣壳。CA16的VP1,VP2,VP3可以构成其VLP。本部分实验我们提取CA16的基因组,通过特异性引物利用PCR的方法调取CA16 衣壳蛋白 VP1,VP2,VP3 的基因。分别与 pET28a,pET43.1a,pCOLD-SUMO,pMAL-P2X与pMAL-C2X五种质粒进行重组质粒的构建。然后将测序正确的重组质粒在工程大肠杆菌中表达,进行表达条件的摸索。采用最佳的表达条件对VLP进行原核表达,通过亲和层析,分子筛等方法来进行蛋白纯化。利用western blot和电子显微镜对所获得的VLPs进行鉴定。
[Abstract]:Enterococcus faecalis is a normal flora of human or animal intestines, oral and reproductive tract. Under normal circumstances, the symbiotic Enterococcus has no pathogenicity to the host. But when it is ectopic parasitism, it can cause septicemia, urinary tract infection, pyogenic abdominal infection and endocarditis. With the increasing resistance of Enterococcus faecalis, the infection is more difficult. The emergence of the bacteriophage has provided us with a new way of thinking. The study of phage and phage lysase has also shown great application value. Scientists have been from various pathogenic bacteria infected by phage, including Streptococcus pneumoniae, anthrax, Lactobacillus, Staphylococcus aureus and so on. The bacteriophage was separated. But with the emergence of antibiotics, people's attention shifted to antibiotics and ignored the bacterial natural enemy phage. With the overstrength of the pathogenic bacteria, the problem of broad-spectrum resistance made phage therapy to be used in the treatment of antibiotic resistant bacterial infections. The heterosexual lysis bacteria, which do not infect eukaryotes or destroy the normal flora of the body, are the antibacterial substances of the new concept. In the deep study of phage and its lysase, a new type of antibacterial agent will be developed for the treatment of bacterial diseases. In the early experiments, we isolated the fecal intestines of vancomycin from the clinical samples. The lysate gene of the bacteriophage was determined by the whole genome sequencing and analysis. By gene engineering expression, 36 faecal Enterococcus was selected from the lylyase LysN10. experiment, and 32 of them could be cracked by LysN10, and the CHAP fragment of LysN10 and LysGH had a certain amino acid sequence. The lysase showed broad-spectrum and efficient lysis activity to Enterococcus faecalis, showing the potential of the lysase for the treatment of antibiotic resistant enterococcus infection. In order to analyze the structure of bacteriophage lyase LysN10 by crystal diffraction, this paper first obtained the prokaryotic expression vector of pET15b-N10 and expressed it in Escherichia coli. The target protein with purity of 95% was obtained by means of conditions, affinity chromatography, molecular sieve chromatography and so on. For the full length LysN10, we screened the crystal conditions by the protein crystallization condition screening kit and failed to obtain the protein crystal through a continuous month of observation. We speculate that the lysase is composed of two independent domains, between the domains through a long flexible linker connection. This structure is difficult to crystallize. In order to solve this problem, we use the homologous protein structure analysis method, through sequence analysis, from the binding domain and functional domain to the whole. The long protein design was truncated and 3 truncated mutants were constructed, named NF418, NR438 and NR492. to express, purify and optimize the constructed recombinant plasmids pET15b-NF418, pET15b-NR438 and pET15b-NR492 respectively. By screening the 296 crystallization conditions, we finally obtained the mutant NF41 8 of the crystal. This lays the foundation for the complete analysis of the structure of LysN10. Hand (foot and mouth disease, HFMD) is an infectious disease caused by enterovirus. The Coxsackie virus A group 16 (CoxsackievirusA16, CA16) is one of the major members of CV. In 1951, the symptoms of hand foot and mouth disease caused by the successful isolation of.CA16 were relative to the first in South Africa. Light, and the specific symptoms of hand foot and mouth disease caused by EV71 is difficult to distinguish, in addition, the children and the recessive infected people are the source of infection, adult infection becomes recessive and asymptomatic virus, it is not ill, but can be used as a potential source of infection to infect children, which is also the main cause of the disease spread difficult to control. 1. Many viral structural proteins have the ability to automatically assemble into VLPs, similar to natural virus particles in morphological structure, and have strong immunogenicity and biological activity. Because VLPs does not contain viral genetic material, it is not infective, some of which have been successfully applied to the clinic as a result of the VLPs surface. Repetitive and high-density display of exogenous epitopes thus triggering a strong immune response, so it is an ideal form of development for a variety of diseases. Research shows that CA16 virus is a subunit consisting of 60 VP1, VP2, VP3, and VP4 capsid proteins together into a twenty - hedral spherical particle. And other small RNA The virus is the same, its VP1, VP2, and VP3 proteins each form a subunit of the capsid. The subunit forms a five polymer, and the 12 five polymer eventually forms the VP1 of the capsid.CA16, and the VP3 can form the VLP. part of the experiment. We extract the genome of CA16 and take the CA16 capsid protein VP1 by specific primers by PCR. The recombinant plasmid was constructed with five plasmids of pET28a, pET43.1a, pCOLD-SUMO, pMAL-P2X and pMAL-C2X respectively. Then the correct recombinant plasmid was expressed in engineering Escherichia coli, and the expression conditions were carried out. The best expression conditions were used to prokaryotic expression of VLP, through affinity chromatography, molecular sieve and other methods. Protein was purified. The VLPs obtained was identified by Western blot and electron microscope.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R37

【参考文献】