本文主要研究内容
作者叶华（2019）在《脂多糖广谱型核酸适配体的定向筛选、序列优化及分析应用》一文中研究指出：脂多糖(Lipopolysaccharide,LPS)是革兰氏阴性细菌细胞膜的固有组成成分,是对宿主具有一定生物毒性的非分泌性物质,所以又称内毒素(Endotoxin)。当细菌裂解或死亡,脂多糖便释放出来,若进入血液则能引起机体发热,产生一系列的炎症反应,是败血性休克、多器官功能障碍综合症等疾病发生的主要致病因子。脂多糖污染不可避免,常见于食品及饮用水、药品及医疗产品中,对人体健康产生巨大的威胁和危害。因此,筛选特异性的脂多糖识别分子和拮抗物质一直是人们关注的重点和研究热点。核酸适配体(Aptamer)因具有亲和力高、特异性强、易于修饰、体外合成方便、不需要动物实验等特点已成为近年来生物识别分子领域的研究热点,在安全检测、疾病治疗、生物分析等方面得到了广泛的应用。本研究采用Capture-SELEX技术并加以改进,以鼠伤寒沙门氏菌脂多糖为靶标筛选获得了一条广谱型脂多糖核酸适配体,然后对其序列结构进行剪裁优化,同时结合荧光偏振、圆二色谱、等温滴定微量热以及分子模拟等技术探讨了序列结构功能,分析了核酸适配体与脂多糖的结合机制,建立了快速、均相的检测方法并进行了细胞抗炎性能试验,为今后脂多糖的快速检测及拮抗分子的开发奠定了良好的基础。具体研究内容如下:首先,基于Capture-SELEX技术建立定向策略筛选获得了广谱型脂多糖核酸适配体。根据碱基互补配对原则设计了一条互补序列作为桥接序列,将ssDNA筛选文库固定在合成的磁性纳米颗粒上,然后将筛选靶标鼠伤寒沙门氏菌脂多糖与其孵育,这样有亲和力、特异性的ssDNA从磁珠上解离下来与靶标形成游离复合物,通过磁分离获得PCR模板进行扩增、酶切制备单链后进行下一轮循环,经过前6轮的正筛、反筛富集后,引入鼠伤寒沙门氏菌等活菌作为定向筛选物质,再经过9轮的正筛、反筛和定向筛交替筛选后,进行克隆测序以及亲和力、特异性试验,最终获得了一条能识别四种细菌来源脂多糖的广谱型核酸适配体EA7,其K_d值为102±17 nM。其次,为进一步验证EA7分子的识别性能,构建了Structure-switching型核酸适配体生物传感器,实现了脂多糖的均相检测。在5’端标记FAM荧光基团的EA7序列的上游设计了一条3’端带有DABCYL猝灭基团标记的短互补序列qCS,在没有靶标脂多糖存在的情况下,核酸适配体与qCS结合,使得FAM与DABCYL相互靠近发生荧光共振能量转移,荧光被猝灭;当靶标出现后,由于特异性亲和力作用,脂多糖取代qCS与EA7结合,而使EA7结构发生转变,从而互补链qCS从EA7上解离下来,FAM与DABCYL相互远离,荧光恢复,根据荧光变化可实现LPS的均相检测。在最优条件下,对鼠伤寒沙门氏菌脂多糖、肠炎沙门氏菌脂多糖、铜绿假单胞菌脂多糖和大肠杆菌脂多糖线性范围分别为:5-250 ng/mL、0.025-10?g/mL、0.05-5?g/mL、0.1-10?g/mL,最低检测限分别为3.0 ng/mL、21.30 ng/mL、47.70 ng/mL、99.31 ng/mL,饮用水加标回收率在95.1%-105.1%之间。该方法操作简便、无需分离、灵敏可靠。第三,对脂多糖广谱型核酸适配体EA7(为便于编号,重新命名为LA80)序列结构及功能进行了剪裁优化和分析。根据核酸适配体的一级序列和二级序列结构对全长核酸适配体LA80采用两种剪裁优化策略,共获得7条剪裁序列,然后构建了荧光偏振竞争法对其进行特异性分析以及利用ITC法进行亲和性分析,最终通过优化去除了部分多余的序列,确定长27 bp的核酸适配体LA27为最佳优化核酸适配体,它不仅保留了对鼠伤寒沙门氏菌脂多糖、铜绿假单胞菌脂多糖和大肠杆菌脂多糖等3种脂多糖的结合能力,其亲和力还提高了约1倍,解离常数K_d=46.2±9.5 nM。通过分析,我们认为LA27上5’端的CC和3’端CGA形成的开环状部分可能是脂多糖结合的支撑区,而其余碱基组成的茎环结构则可能是与脂多糖发生作用的主要结合区。圆二色谱CD结果进一步表明LA27可能以嵌入的方式与脂多糖结合。第四,建立了基于氧化石墨烯(GO)增强荧光偏振信号的分析方法快速检测脂多糖,以对LA27在分析检测方面的性能进一步进行分析。利用水浴超声法制备获得的氧化石墨烯纳米片吸附核酸适配体分子探针,形成GO/aptamer复合物,荧光偏振信号迅速增强,加入脂多糖后,分子探针从GO上解离下来与其结合形成LPS/aptamer复合物,荧光偏振信号降低,从而实现了脂多糖的快速、均相检测。结果表明,在最优条件下,LA27对鼠伤寒沙门氏菌脂多糖、铜绿假单胞菌脂多糖和大肠杆菌脂多糖的检测限分别为38.7 ng/mL、88.7 ng/mL、154 ng/mL,比LA80作为分子探针的检测限分别提高了4.8、29和18倍;动力学试验结果表明该方法检测快速,可在30 min内完成检测。氯化钠注射液样品加标回收率在92%-112.5%之间。以上结果表明剪裁优化后的LA27由于序列更短、亲和力更好,使其检测效果更佳。第五,分析探讨了核酸适配体LA27的抗炎性能及其与LPS的结合位点和作用模式。以四种脂多糖分别诱导HepG2细胞使其产生炎症因子,利用ELISA法检测了空白组、LPS诱导组、LA27抑制组中TNF-?,IL-1?和IL-6等三种炎症因子的表达水平。结果发现,LA27抑制组中由鼠伤寒沙门氏菌脂多糖、铜绿假单胞菌脂多糖和大肠杆菌脂多糖三种脂多糖分别刺激的细胞中TNF-?,IL-1?和IL-6炎症因子的表达水平显著降低(p<0.01),而肠炎沙门氏菌脂多糖中表达的炎症因子水平无显著性差异(p<0.01)。这说明LA27具有一定的抗炎效果,且进一步验证了其对鼠伤寒沙门氏菌脂多糖、铜绿假单胞菌脂多糖和大肠杆菌脂多糖的识别能力。利用MOE2018软件模拟分析发现,在疏水作用、氢键作用以及静电作用三种力的共同作用下,LPS可能通过类脂A上的脂肪酸链与由LA27上T3、T4、T6、T16、T17、T19、T20等7个胸腺嘧啶T碱基形成的疏水性大沟区域结合,形成了稳定的类似“T”型结构的复合物,从而特异性识别。
Abstract
zhi duo tang (Lipopolysaccharide,LPS)shi ge lan shi yin xing xi jun xi bao mo de gu you zu cheng cheng fen ,shi dui su zhu ju you yi ding sheng wu du xing de fei fen bi xing wu zhi ,suo yi you chen nei du su (Endotoxin)。dang xi jun lie jie huo si wang ,zhi duo tang bian shi fang chu lai ,re jin ru xie ye ze neng yin qi ji ti fa re ,chan sheng yi ji lie de yan zheng fan ying ,shi bai xie xing xiu ke 、duo qi guan gong neng zhang ai zeng ge zheng deng ji bing fa sheng de zhu yao zhi bing yin zi 。zhi duo tang wu ran bu ke bi mian ,chang jian yu shi pin ji yin yong shui 、yao pin ji yi liao chan pin zhong ,dui ren ti jian kang chan sheng ju da de wei xie he wei hai 。yin ci ,shai shua te yi xing de zhi duo tang shi bie fen zi he jie kang wu zhi yi zhi shi ren men guan zhu de chong dian he yan jiu re dian 。he suan kuo pei ti (Aptamer)yin ju you qin he li gao 、te yi xing jiang 、yi yu xiu shi 、ti wai ge cheng fang bian 、bu xu yao dong wu shi yan deng te dian yi cheng wei jin nian lai sheng wu shi bie fen zi ling yu de yan jiu re dian ,zai an quan jian ce 、ji bing zhi liao 、sheng wu fen xi deng fang mian de dao le an fan de ying yong 。ben yan jiu cai yong Capture-SELEXji shu bing jia yi gai jin ,yi shu shang han sha men shi jun zhi duo tang wei ba biao shai shua huo de le yi tiao an pu xing zhi duo tang he suan kuo pei ti ,ran hou dui ji xu lie jie gou jin hang jian cai you hua ,tong shi jie ge ying guang pian zhen 、yuan er se pu 、deng wen di ding wei liang re yi ji fen zi mo ni deng ji shu tan tao le xu lie jie gou gong neng ,fen xi le he suan kuo pei ti yu zhi duo tang de jie ge ji zhi ,jian li le kuai su 、jun xiang de jian ce fang fa bing jin hang le xi bao kang yan xing neng shi yan ,wei jin hou zhi duo tang de kuai su jian ce ji jie kang fen zi de kai fa dian ding le liang hao de ji chu 。ju ti yan jiu nei rong ru xia :shou xian ,ji yu Capture-SELEXji shu jian li ding xiang ce lve shai shua huo de le an pu xing zhi duo tang he suan kuo pei ti 。gen ju jian ji hu bu pei dui yuan ze she ji le yi tiao hu bu xu lie zuo wei qiao jie xu lie ,jiang ssDNAshai shua wen ku gu ding zai ge cheng de ci xing na mi ke li shang ,ran hou jiang shai shua ba biao shu shang han sha men shi jun zhi duo tang yu ji fu yo ,zhe yang you qin he li 、te yi xing de ssDNAcong ci zhu shang jie li xia lai yu ba biao xing cheng you li fu ge wu ,tong guo ci fen li huo de PCRmo ban jin hang kuo zeng 、mei qie zhi bei chan lian hou jin hang xia yi lun xun huan ,jing guo qian 6lun de zheng shai 、fan shai fu ji hou ,yin ru shu shang han sha men shi jun deng huo jun zuo wei ding xiang shai shua wu zhi ,zai jing guo 9lun de zheng shai 、fan shai he ding xiang shai jiao ti shai shua hou ,jin hang ke long ce xu yi ji qin he li 、te yi xing shi yan ,zui zhong huo de le yi tiao neng shi bie si chong xi jun lai yuan zhi duo tang de an pu xing he suan kuo pei ti EA7,ji K_dzhi wei 102±17 nM。ji ci ,wei jin yi bu yan zheng EA7fen zi de shi bie xing neng ,gou jian le Structure-switchingxing he suan kuo pei ti sheng wu chuan gan qi ,shi xian le zhi duo tang de jun xiang jian ce 。zai 5’duan biao ji FAMying guang ji tuan de EA7xu lie de shang you she ji le yi tiao 3’duan dai you DABCYLcu mie ji tuan biao ji de duan hu bu xu lie qCS,zai mei you ba biao zhi duo tang cun zai de qing kuang xia ,he suan kuo pei ti yu qCSjie ge ,shi de FAMyu DABCYLxiang hu kao jin fa sheng ying guang gong zhen neng liang zhuai yi ,ying guang bei cu mie ;dang ba biao chu xian hou ,you yu te yi xing qin he li zuo yong ,zhi duo tang qu dai qCSyu EA7jie ge ,er shi EA7jie gou fa sheng zhuai bian ,cong er hu bu lian qCScong EA7shang jie li xia lai ,FAMyu DABCYLxiang hu yuan li ,ying guang hui fu ,gen ju ying guang bian hua ke shi xian LPSde jun xiang jian ce 。zai zui you tiao jian xia ,dui shu shang han sha men shi jun zhi duo tang 、chang yan sha men shi jun zhi duo tang 、tong lu jia chan bao jun zhi duo tang he da chang gan jun zhi duo tang xian xing fan wei fen bie wei :5-250 ng/mL、0.025-10?g/mL、0.05-5?g/mL、0.1-10?g/mL,zui di jian ce xian fen bie wei 3.0 ng/mL、21.30 ng/mL、47.70 ng/mL、99.31 ng/mL,yin yong shui jia biao hui shou lv zai 95.1%-105.1%zhi jian 。gai fang fa cao zuo jian bian 、mo xu fen li 、ling min ke kao 。di san ,dui zhi duo tang an pu xing he suan kuo pei ti EA7(wei bian yu bian hao ,chong xin ming ming wei LA80)xu lie jie gou ji gong neng jin hang le jian cai you hua he fen xi 。gen ju he suan kuo pei ti de yi ji xu lie he er ji xu lie jie gou dui quan chang he suan kuo pei ti LA80cai yong liang chong jian cai you hua ce lve ,gong huo de 7tiao jian cai xu lie ,ran hou gou jian le ying guang pian zhen jing zheng fa dui ji jin hang te yi xing fen xi yi ji li yong ITCfa jin hang qin he xing fen xi ,zui zhong tong guo you hua qu chu le bu fen duo yu de xu lie ,que ding chang 27 bpde he suan kuo pei ti LA27wei zui jia you hua he suan kuo pei ti ,ta bu jin bao liu le dui shu shang han sha men shi jun zhi duo tang 、tong lu jia chan bao jun zhi duo tang he da chang gan jun zhi duo tang deng 3chong zhi duo tang de jie ge neng li ,ji qin he li hai di gao le yao 1bei ,jie li chang shu K_d=46.2±9.5 nM。tong guo fen xi ,wo men ren wei LA27shang 5’duan de CChe 3’duan CGAxing cheng de kai huan zhuang bu fen ke neng shi zhi duo tang jie ge de zhi cheng ou ,er ji yu jian ji zu cheng de jing huan jie gou ze ke neng shi yu zhi duo tang fa sheng zuo yong de zhu yao jie ge ou 。yuan er se pu CDjie guo jin yi bu biao ming LA27ke neng yi qian ru de fang shi yu zhi duo tang jie ge 。di si ,jian li le ji yu yang hua dan mo xi (GO)zeng jiang ying guang pian zhen xin hao de fen xi fang fa kuai su jian ce zhi duo tang ,yi dui LA27zai fen xi jian ce fang mian de xing neng jin yi bu jin hang fen xi 。li yong shui yu chao sheng fa zhi bei huo de de yang hua dan mo xi na mi pian xi fu he suan kuo pei ti fen zi tan zhen ,xing cheng GO/aptamerfu ge wu ,ying guang pian zhen xin hao xun su zeng jiang ,jia ru zhi duo tang hou ,fen zi tan zhen cong GOshang jie li xia lai yu ji jie ge xing cheng LPS/aptamerfu ge wu ,ying guang pian zhen xin hao jiang di ,cong er shi xian le zhi duo tang de kuai su 、jun xiang jian ce 。jie guo biao ming ,zai zui you tiao jian xia ,LA27dui shu shang han sha men shi jun zhi duo tang 、tong lu jia chan bao jun zhi duo tang he da chang gan jun zhi duo tang de jian ce xian fen bie wei 38.7 ng/mL、88.7 ng/mL、154 ng/mL,bi LA80zuo wei fen zi tan zhen de jian ce xian fen bie di gao le 4.8、29he 18bei ;dong li xue shi yan jie guo biao ming gai fang fa jian ce kuai su ,ke zai 30 minnei wan cheng jian ce 。lv hua na zhu she ye yang pin jia biao hui shou lv zai 92%-112.5%zhi jian 。yi shang jie guo biao ming jian cai you hua hou de LA27you yu xu lie geng duan 、qin he li geng hao ,shi ji jian ce xiao guo geng jia 。di wu ,fen xi tan tao le he suan kuo pei ti LA27de kang yan xing neng ji ji yu LPSde jie ge wei dian he zuo yong mo shi 。yi si chong zhi duo tang fen bie you dao HepG2xi bao shi ji chan sheng yan zheng yin zi ,li yong ELISAfa jian ce le kong bai zu 、LPSyou dao zu 、LA27yi zhi zu zhong TNF-?,IL-1?he IL-6deng san chong yan zheng yin zi de biao da shui ping 。jie guo fa xian ,LA27yi zhi zu zhong you shu shang han sha men shi jun zhi duo tang 、tong lu jia chan bao jun zhi duo tang he da chang gan jun zhi duo tang san chong zhi duo tang fen bie ci ji de xi bao zhong TNF-?,IL-1?he IL-6yan zheng yin zi de biao da shui ping xian zhe jiang di (p<0.01),er chang yan sha men shi jun zhi duo tang zhong biao da de yan zheng yin zi shui ping mo xian zhe xing cha yi (p<0.01)。zhe shui ming LA27ju you yi ding de kang yan xiao guo ,ju jin yi bu yan zheng le ji dui shu shang han sha men shi jun zhi duo tang 、tong lu jia chan bao jun zhi duo tang he da chang gan jun zhi duo tang de shi bie neng li 。li yong MOE2018ruan jian mo ni fen xi fa xian ,zai shu shui zuo yong 、qing jian zuo yong yi ji jing dian zuo yong san chong li de gong tong zuo yong xia ,LPSke neng tong guo lei zhi Ashang de zhi fang suan lian yu you LA27shang T3、T4、T6、T16、T17、T19、T20deng 7ge xiong xian mi ding Tjian ji xing cheng de shu shui xing da gou ou yu jie ge ,xing cheng le wen ding de lei shi “T”xing jie gou de fu ge wu ,cong er te yi xing shi bie 。
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论文作者分别是来自江南大学的叶华，发表于刊物江南大学2019-10-21论文，是一篇关于脂多糖核酸适配体论文,定向筛选论文,剪裁优化论文,荧光偏振论文,分子模拟论文,江南大学2019-10-21论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自江南大学2019-10-21论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：脂多糖核酸适配体论文; 定向筛选论文; 剪裁优化论文; 荧光偏振论文; 分子模拟论文; 江南大学2019-10-21论文;