本文選題：糞腸球菌 + 噬菌體裂解酶　； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：糞腸球菌是人或動(dòng)物腸道、口腔和生殖道的正常菌群,正常情況下,共生的糞腸球菌對(duì)宿主沒(méi)有致病性。但當(dāng)其異位寄生時(shí)可引起敗血癥、尿路感染、化膿性腹部感染和心內(nèi)膜炎等疾病,隨著糞腸球菌耐藥性加重,其所引起的感染更加難以控制,迫切需要研究新型抗菌制劑。噬菌體的出現(xiàn)為我們提供了一個(gè)新的思路,對(duì)于噬菌體及噬菌體裂解酶的研究也顯現(xiàn)出巨大的應(yīng)用價(jià)值�？茖W(xué)家從各種感染了噬菌體的致病菌,包括肺炎鏈球菌、炭疽桿菌、乳酸桿菌、金黃葡萄球菌等都分離出噬菌體。但隨著抗生素的出現(xiàn),人們的注意力又轉(zhuǎn)移到抗生素上面來(lái),從而忽略了細(xì)菌的天敵噬菌體。隨著病原細(xì)菌的超強(qiáng)、廣譜耐藥性問(wèn)題使得噬菌體療法用于耐藥性細(xì)菌感染的治療成為研究的熱點(diǎn)。噬菌體及其裂解酶可以特異性的裂解細(xì)菌,不會(huì)侵染真核細(xì)胞,也不會(huì)破壞機(jī)體的正常菌群,是新概念的抗菌物質(zhì)。對(duì)噬菌體及其裂解酶的深入研究,將會(huì)開(kāi)發(fā)出新型抗菌制劑,用于細(xì)菌性疾病的治療。前期實(shí)驗(yàn)中,我們由臨床樣本中分離得到了對(duì)耐萬(wàn)古霉素糞腸球菌具有高效裂解活性的新噬菌體,通過(guò)全基因組序列測(cè)定和分析,確定了噬菌體的裂解酶基因,通過(guò)基因工程表達(dá)獲得了裂解酶LysN10。實(shí)驗(yàn)共選取36株糞腸球菌,LysN10可以裂解其中32株菌,且LysN10與LysGH的CHAP片段在氨基酸序列具有一定的同源性。裂解酶對(duì)糞腸球菌表現(xiàn)出廣譜、高效的裂解活性,顯示了該裂解酶治療耐藥性糞腸球菌感染的潛力。為通過(guò)晶體衍射方法解析噬菌體裂解酶LysN10的結(jié)構(gòu),本論文首先獲得了pET15b-N10的原核表達(dá)載體,并于大腸桿菌中表達(dá)。通過(guò)表達(dá)條件的摸索、親和層析、分子篩層析等方法得到了純度達(dá)95%的目標(biāo)蛋白。對(duì)于全長(zhǎng)LysN10我們采用蛋白質(zhì)結(jié)晶條件篩選試劑盒進(jìn)行晶體結(jié)晶條件的篩選,通過(guò)連續(xù)一個(gè)月的觀察,未能獲得蛋白晶體。通過(guò)對(duì)裂解酶和同源蛋白結(jié)構(gòu)的分析,我們推測(cè)該裂解酶由兩個(gè)獨(dú)立的結(jié)構(gòu)域組成,結(jié)構(gòu)域間通過(guò)一個(gè)較長(zhǎng)的柔性linker連接。這樣的結(jié)構(gòu)特點(diǎn),造成該全長(zhǎng)蛋白很難結(jié)晶。為解決這一問(wèn)題,我們借鑒同源蛋白結(jié)構(gòu)解析方式,通過(guò)序列分析,分別從結(jié)合結(jié)構(gòu)域和功能結(jié)構(gòu)域?qū)θL(zhǎng)蛋白設(shè)計(jì)截短,構(gòu)建了 3個(gè)不同位點(diǎn)的截?cái)嗤蛔凅w,命名為NF418,NR438和NR492。對(duì)已經(jīng)構(gòu)建好的重組質(zhì)粒pET15b-NF418,pET15b-NR438和pET15b-NR492分別進(jìn)行表達(dá)、純化以及結(jié)晶條件的篩選和優(yōu)化。通過(guò)對(duì)296種結(jié)晶條件的篩選,我們最終獲得突變體NF418的晶體。這為完全解析LysN10的結(jié)構(gòu)奠定基礎(chǔ)。手足口病(hand,foot and mouth disease,HFMD)是由腸道病毒引起的一種傳染病,柯薩奇病毒A組16型(CoxsackievirusA16,CA16)是CV的主要成員之一,1951年首次在南非被成功分離。CA16所致的手足口病癥狀相對(duì)較輕,且與由EV71引起的手足口病的具體癥狀難以區(qū)別,另外,患兒和隱性感染者均為傳染源,成人感染后成為隱性感染者和無(wú)癥狀帶毒者,本身不發(fā)病,但可作為潛在的傳染源將病毒傳染給幼兒,這也是造成疾病傳播難以控制的主要原因之一。許多病毒結(jié)構(gòu)蛋白都具有自動(dòng)組裝成VLPs的能力,在形態(tài)結(jié)構(gòu)上與天然的病毒顆粒相似,具有很強(qiáng)的免疫原性和生物學(xué)活性。由于VLPs不含有病毒遺傳物質(zhì),因此不具有感染性,其中有些已經(jīng)作為疫苗成功應(yīng)用于臨床。由于VLPs表面能夠重復(fù)且高密度展示外源性表位從而引發(fā)強(qiáng)有力的免疫應(yīng)答,因此它對(duì)于多種疾病來(lái)說(shuō)都是一種可開(kāi)發(fā)理想的疫苗形式。研究表明,CA16病毒是由60個(gè)VP1,VP2,VP3以及VP4衣殼蛋白構(gòu)成的亞單位共同形成一個(gè)二十面體的球形顆粒。與其他小RNA病毒相同,其VP1,VP2,和VP3蛋白各自組成衣殼的一個(gè)亞單位,此亞單位又構(gòu)成一個(gè)五聚體,12個(gè)五聚體最終構(gòu)成衣殼。CA16的VP1,VP2,VP3可以構(gòu)成其VLP。本部分實(shí)驗(yàn)我們提取CA16的基因組,通過(guò)特異性引物利用PCR的方法調(diào)取CA16 衣殼蛋白 VP1,VP2,VP3 的基因。分別與 pET28a,pET43.1a,pCOLD-SUMO,pMAL-P2X與pMAL-C2X五種質(zhì)粒進(jìn)行重組質(zhì)粒的構(gòu)建。然后將測(cè)序正確的重組質(zhì)粒在工程大腸桿菌中表達(dá),進(jìn)行表達(dá)條件的摸索。采用最佳的表達(dá)條件對(duì)VLP進(jìn)行原核表達(dá),通過(guò)親和層析,分子篩等方法來(lái)進(jìn)行蛋白純化。利用western blot和電子顯微鏡對(duì)所獲得的VLPs進(jìn)行鑒定。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R37

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