發(fā)布時間：2018-06-16 16:10

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

【摘要】：糞腸球菌是人或動物腸道、口腔和生殖道的正常菌群,正常情況下,共生的糞腸球菌對宿主沒有致病性。但當其異位寄生時可引起敗血癥、尿路感染、化膿性腹部感染和心內膜炎等疾病,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。噬菌體的出現為我們提供了一個新的思路,對于噬菌體及噬菌體裂解酶的研究也顯現出巨大的應用價值�？茖W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,人們的注意力又轉移到抗生素上面來,從而忽略了細菌的天敵噬菌體。隨著病原細菌的超強、廣譜耐藥性問題使得噬菌體療法用于耐藥性細菌感染的治療成為研究的熱點。噬菌體及其裂解酶可以特異性的裂解細菌,不會侵染真核細胞,也不會破壞機體的正常菌群,是新概念的抗菌物質。對噬菌體及其裂解酶的深入研究,將會開發(fā)出新型抗菌制劑,用于細菌性疾病的治療。前期實驗中,我們由臨床樣本中分離得到了對耐萬古霉素糞腸球菌具有高效裂解活性的新噬菌體,通過全基因組序列測定和分析,確定了噬菌體的裂解酶基因,通過基因工程表達獲得了裂解酶LysN10。實驗共選取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。裂解酶對糞腸球菌表現出廣譜、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。為通過晶體衍射方法解析噬菌體裂解酶LysN10的結構,本論文首先獲得了pET15b-N10的原核表達載體,并于大腸桿菌中表達。通過表達條件的摸索、親和層析、分子篩層析等方法得到了純度達95%的目標蛋白。對于全長LysN10我們采用蛋白質結晶條件篩選試劑盒進行晶體結晶條件的篩選,通過連續(xù)一個月的觀察,未能獲得蛋白晶體。通過對裂解酶和同源蛋白結構的分析,我們推測該裂解酶由兩個獨立的結構域組成,結構域間通過一個較長的柔性linker連接。這樣的結構特點,造成該全長蛋白很難結晶。為解決這一問題,我們借鑒同源蛋白結構解析方式,通過序列分析,分別從結合結構域和功能結構域對全長蛋白設計截短,構建了 3個不同位點的截斷突變體,命名為NF418,NR438和NR492。對已經構建好的重組質粒pET15b-NF418,pET15b-NR438和pET15b-NR492分別進行表達、純化以及結晶條件的篩選和優(yōu)化。通過對296種結晶條件的篩選,我們最終獲得突變體NF418的晶體。這為完全解析LysN10的結構奠定基礎。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。許多病毒結構蛋白都具有自動組裝成VLPs的能力,在形態(tài)結構上與天然的病毒顆粒相似,具有很強的免疫原性和生物學活性。由于VLPs不含有病毒遺傳物質,因此不具有感染性,其中有些已經作為疫苗成功應用于臨床。由于VLPs表面能夠重復且高密度展示外源性表位從而引發(fā)強有力的免疫應答,因此它對于多種疾病來說都是一種可開發(fā)理想的疫苗形式。研究表明,CA16病毒是由60個VP1,VP2,VP3以及VP4衣殼蛋白構成的亞單位共同形成一個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亞單位,此亞單位又構成一個五聚體,12個五聚體最終構成衣殼。CA16的VP1,VP2,VP3可以構成其VLP。本部分實驗我們提取CA16的基因組,通過特異性引物利用PCR的方法調取CA16 衣殼蛋白 VP1,VP2,VP3 的基因。分別與 pET28a,pET43.1a,pCOLD-SUMO,pMAL-P2X與pMAL-C2X五種質粒進行重組質粒的構建。然后將測序正確的重組質粒在工程大腸桿菌中表達,進行表達條件的摸索。采用最佳的表達條件對VLP進行原核表達,通過親和層析,分子篩等方法來進行蛋白純化。利用western blot和電子顯微鏡對所獲得的VLPs進行鑒定。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R37

【參考文獻】

相關期刊論文 前10條

1 查杰;馬智龍;周純葆;;引起兩起手足口病聚集性疫情的柯薩奇病毒A6的VP1基因特征及其分子流行病學研究[J];中國衛(wèi)生檢驗雜志;2015年02期

2 謝席平;黃蓮珍;劉讓萬;何聲福;陳華云;陳嘉昌;湯鏈;;檢測柯薩奇病毒A組6、10、16型的多重熒光RT-PCR建立及應用[J];臨床檢驗雜志;2014年10期

3 陳祥鵬;檀曉娟;許文波;;柯薩奇病毒A組16型分子流行病學和疫苗研究進展[J];病毒學報;2014年04期

4 馬巖;李麗紅;孫利偉;;對2013年長春市柯薩奇病毒A6型手足口病的臨床分析[J];當代醫(yī)藥論叢;2014年11期

5 陳祥鵬;檀曉娟;張勇;許文波;;柯薩奇病毒A組16型滅活抗原對小鼠免疫保護作用的研究[J];病毒學報;2014年03期

6 楊國梁;馬超鋒;陳海龍;李焱;李一為;杜全麗;李恒新;顏虹;;2013年西安地區(qū)柯薩奇病毒A6流行情況及基因特征[J];西安交通大學學報(醫(yī)學版);2014年04期

7 曹桂華;林琳;劉渠;李剛;劉鳳仁;葉偉雄;陳嘉慧;覃佩蘭;葉碧莉;董建;陳應堅;甘莉萍;徐亞軍;石婷;;2008-2011年廣東省深圳市龍崗區(qū)手足口病流行病學特征分析[J];疾病監(jiān)測;2012年11期

8 辛娜;井發(fā)紅;李敬梅;尤濤;;醫(yī)院感染革蘭氏陽性球菌的分布與耐藥性分析[J];國際檢驗醫(yī)學雜志;2011年18期

9 張偉;王玉光;楊朝暉;龐保東;吳昊;楊巧芝;金敏;楊金玲;李興旺;劉清泉;張永利;;腸道病毒71型與柯薩奇A組16型混合感染致手足口病并發(fā)中樞神經系統(tǒng)感染臨床分析[J];中國全科醫(yī)學;2011年29期

10 劉靜;楊靖嫻;王樹琴;王志娟;梁國威;;2007～2010年糞腸球菌、屎腸球菌耐藥性變遷分析[J];國際檢驗醫(yī)學雜志;2011年08期

，

本文編號：2027281