本文主要研究内容
作者易夏玉（2019）在《LTA模拟肽疫苗诱导免疫应答并保护小鼠抵御金葡菌感染》一文中研究指出：金黄色葡萄球菌是社区和院内感染的主要病原体,可引发菌血症、腹膜炎、肺炎,甚至脓毒症性休克而死亡。脂磷壁酸LTA(lipoteichoicacid)是金葡菌细胞壁的重要保守结构,对金葡菌的生长分裂,宿主细胞黏附等具有重要作用。但由于LTA属于是胸腺非依赖抗原,不易诱导免疫应答及免疫记忆,因此不适于作为疫苗候选。研究表明利用噬菌体展示技术可获得模拟多糖的表位序列。在前期工作中我们利用该技术得到多个模拟LTA表位的序列,根据其中一个阳性序列合成四分枝多价抗原肽(命名为MAP2-3)。MAP2-3免疫后可保护小鼠抵御金葡菌系统性感染,包括在脓毒症休克模型中提高小鼠生存率以及在菌血症模型中降低脏器中细菌载量。本部分工作进一步探讨MAP2-3免疫诱导保护作用的机制。主要包含以下内容:第一部分LTA模拟肽的合成及其抗血清特异性的鉴定1.根据前期筛选所得阳性克隆序列,通过在线软件预测序列理化特性,分析表明阳性序列GHKEDRQWCQHS的亲水性、可及性均高于另外两条序列;根据该序列合成四份支多价抗原肽(称为MAP2-3)。ELISA结果表明,MAP2-3能与两种不同的抗LTA单抗结合;2.以MAP2-3或MAP支架(MAPctrl)加弗氏佐剂(按体积比1:1)乳化,免疫BALB/c鼠(100μg/只,皮下注射),两次免疫间隔2周,共4次。第三组小鼠为空白对照组;3.以MAP2-3免疫小鼠。末次免疫后7天采血、收集血清。ELISA结果显示,与MAPctrl组和空白对照组相比,抗MAP2-3血清能特异地与MAP2-3、LTA以及金葡菌超声裂解产物结合。4.末次免疫后7天采血、收集血清。ELISA检测MAP2-3免疫诱导体内产生抗LTA的IgG类抗体。第二部分抗LTA模拟肽抗血清的调理吞噬功能及被动免疫保护作用1.以MAP2-3免疫新西兰兔。末次免疫后7天采血、收集血清。ELISA结果显示,与未免疫血清(NRS)相比,兔抗MAP2-3血清能特异地与MAP2-3、LTA以及金葡菌超声裂解产物结合。2.调理吞噬实验结果表明,在补体存在下,鼠抗MAP2-3抗血清能增强小鼠中性粒细胞杀伤S.aureus。在补体存在或补体灭活情况下,兔抗MAP2-3抗血清能增强小鼠中性粒细胞杀伤S.aureus。3.在菌血症模型中,过继转移抗MAP2-3抗血清显著降低小鼠血液、肺脏及肾脏中的细菌残存量;4.在肺炎模型中,过继转移兔抗MAP2-3抗血清显著降低小鼠肺内细菌载量,同时减轻肺内炎性细胞浸润;5.在皮肤脓肿和坏死模型中,过继转移兔抗MAP2-3抗血清显著减小皮肤坏死面积以及降低脓肿部位细菌载量。第三部分LTA模拟肽诱导特异性免疫应答和免疫记忆的探讨以MAP2-3免疫BALB/c鼠。末次免疫一个月后腹腔注射热灭活金葡菌。7天后无菌取小鼠脾脏、淋巴结、股骨和胫骨,获得单细胞悬液。1.ELISPOT结果显示在MAP2-3小鼠脾细胞中,特异性识别LTA、可分泌IgG的抗体分泌细胞(antibody-secreted cells,ASCs)以及特异性针对S.aureus、分泌IgG的ASCs数量显著高于MAPctrl对照组和空白对照组;2.流式细胞术结果显示,与MAPctrl组和空白对照组相比,MAP2-3免疫组小鼠:(1)脾脏和骨髓中浆细胞(B220-CD138+)表达量明显增高;(2)脾脏中GC-B细胞(CD19+CD95+GL7+)表达百分比明显增高;(3)脾脏中滤泡辅助性T细胞(TFH,CD4+CXCR5+PD-1+)表达百分比明显增高;(4)脾脏和骨髓中记忆性B细胞(B220+CD38+IgD-GL7-IgG+)百分比明显增多;(5)脾脏中记忆性B细胞表面CD80表达量明显增高;(6)脾脏和淋巴结中效应型记忆性T细胞(TEM,CD4+CD44+CD62L-)百分比明显增高。3.以LTA及热灭活S.aureus体外刺激脾细胞24 h,ELISA结果显示LTA刺激MAP2-3免疫组脾细胞分泌IL-6,灭活S.aureus刺激MAP2-3免疫组脾细胞分泌IL-2、IFN-γ和IL-6。结论:模拟LTA的四分支多价抗原肽(MAP2-3)能够有效诱导特异性体液免疫应答和免疫记忆,保护BALB/c小鼠抵御S.aureus感染。这一工作为S.aureus疫苗研究提供新的策略。
Abstract
jin huang se pu tao qiu jun shi she ou he yuan nei gan ran de zhu yao bing yuan ti ,ke yin fa jun xie zheng 、fu mo yan 、fei yan ,shen zhi nong du zheng xing xiu ke er si wang 。zhi lin bi suan LTA(lipoteichoicacid)shi jin pu jun xi bao bi de chong yao bao shou jie gou ,dui jin pu jun de sheng chang fen lie ,su zhu xi bao nian fu deng ju you chong yao zuo yong 。dan you yu LTAshu yu shi xiong xian fei yi lai kang yuan ,bu yi you dao mian yi ying da ji mian yi ji yi ,yin ci bu kuo yu zuo wei yi miao hou shua 。yan jiu biao ming li yong shi jun ti zhan shi ji shu ke huo de mo ni duo tang de biao wei xu lie 。zai qian ji gong zuo zhong wo men li yong gai ji shu de dao duo ge mo ni LTAbiao wei de xu lie ,gen ju ji zhong yi ge yang xing xu lie ge cheng si fen zhi duo jia kang yuan tai (ming ming wei MAP2-3)。MAP2-3mian yi hou ke bao hu xiao shu di yu jin pu jun ji tong xing gan ran ,bao gua zai nong du zheng xiu ke mo xing zhong di gao xiao shu sheng cun lv yi ji zai jun xie zheng mo xing zhong jiang di zang qi zhong xi jun zai liang 。ben bu fen gong zuo jin yi bu tan tao MAP2-3mian yi you dao bao hu zuo yong de ji zhi 。zhu yao bao han yi xia nei rong :di yi bu fen LTAmo ni tai de ge cheng ji ji kang xie qing te yi xing de jian ding 1.gen ju qian ji shai shua suo de yang xing ke long xu lie ,tong guo zai xian ruan jian yu ce xu lie li hua te xing ,fen xi biao ming yang xing xu lie GHKEDRQWCQHSde qin shui xing 、ke ji xing jun gao yu ling wai liang tiao xu lie ;gen ju gai xu lie ge cheng si fen zhi duo jia kang yuan tai (chen wei MAP2-3)。ELISAjie guo biao ming ,MAP2-3neng yu liang chong bu tong de kang LTAchan kang jie ge ;2.yi MAP2-3huo MAPzhi jia (MAPctrl)jia fu shi zuo ji (an ti ji bi 1:1)ru hua ,mian yi BALB/cshu (100μg/zhi ,pi xia zhu she ),liang ci mian yi jian ge 2zhou ,gong 4ci 。di san zu xiao shu wei kong bai dui zhao zu ;3.yi MAP2-3mian yi xiao shu 。mo ci mian yi hou 7tian cai xie 、shou ji xie qing 。ELISAjie guo xian shi ,yu MAPctrlzu he kong bai dui zhao zu xiang bi ,kang MAP2-3xie qing neng te yi de yu MAP2-3、LTAyi ji jin pu jun chao sheng lie jie chan wu jie ge 。4.mo ci mian yi hou 7tian cai xie 、shou ji xie qing 。ELISAjian ce MAP2-3mian yi you dao ti nei chan sheng kang LTAde IgGlei kang ti 。di er bu fen kang LTAmo ni tai kang xie qing de diao li tun shi gong neng ji bei dong mian yi bao hu zuo yong 1.yi MAP2-3mian yi xin xi lan tu 。mo ci mian yi hou 7tian cai xie 、shou ji xie qing 。ELISAjie guo xian shi ,yu wei mian yi xie qing (NRS)xiang bi ,tu kang MAP2-3xie qing neng te yi de yu MAP2-3、LTAyi ji jin pu jun chao sheng lie jie chan wu jie ge 。2.diao li tun shi shi yan jie guo biao ming ,zai bu ti cun zai xia ,shu kang MAP2-3kang xie qing neng zeng jiang xiao shu zhong xing li xi bao sha shang S.aureus。zai bu ti cun zai huo bu ti mie huo qing kuang xia ,tu kang MAP2-3kang xie qing neng zeng jiang xiao shu zhong xing li xi bao sha shang S.aureus。3.zai jun xie zheng mo xing zhong ,guo ji zhuai yi kang MAP2-3kang xie qing xian zhe jiang di xiao shu xie ye 、fei zang ji shen zang zhong de xi jun can cun liang ;4.zai fei yan mo xing zhong ,guo ji zhuai yi tu kang MAP2-3kang xie qing xian zhe jiang di xiao shu fei nei xi jun zai liang ,tong shi jian qing fei nei yan xing xi bao jin run ;5.zai pi fu nong zhong he huai si mo xing zhong ,guo ji zhuai yi tu kang MAP2-3kang xie qing xian zhe jian xiao pi fu huai si mian ji yi ji jiang di nong zhong bu wei xi jun zai liang 。di san bu fen LTAmo ni tai you dao te yi xing mian yi ying da he mian yi ji yi de tan tao yi MAP2-3mian yi BALB/cshu 。mo ci mian yi yi ge yue hou fu qiang zhu she re mie huo jin pu jun 。7tian hou mo jun qu xiao shu pi zang 、lin ba jie 、gu gu he jing gu ,huo de chan xi bao xuan ye 。1.ELISPOTjie guo xian shi zai MAP2-3xiao shu pi xi bao zhong ,te yi xing shi bie LTA、ke fen bi IgGde kang ti fen bi xi bao (antibody-secreted cells,ASCs)yi ji te yi xing zhen dui S.aureus、fen bi IgGde ASCsshu liang xian zhe gao yu MAPctrldui zhao zu he kong bai dui zhao zu ;2.liu shi xi bao shu jie guo xian shi ,yu MAPctrlzu he kong bai dui zhao zu xiang bi ,MAP2-3mian yi zu xiao shu :(1)pi zang he gu sui zhong jiang xi bao (B220-CD138+)biao da liang ming xian zeng gao ;(2)pi zang zhong GC-Bxi bao (CD19+CD95+GL7+)biao da bai fen bi ming xian zeng gao ;(3)pi zang zhong lv pao fu zhu xing Txi bao (TFH,CD4+CXCR5+PD-1+)biao da bai fen bi ming xian zeng gao ;(4)pi zang he gu sui zhong ji yi xing Bxi bao (B220+CD38+IgD-GL7-IgG+)bai fen bi ming xian zeng duo ;(5)pi zang zhong ji yi xing Bxi bao biao mian CD80biao da liang ming xian zeng gao ;(6)pi zang he lin ba jie zhong xiao ying xing ji yi xing Txi bao (TEM,CD4+CD44+CD62L-)bai fen bi ming xian zeng gao 。3.yi LTAji re mie huo S.aureusti wai ci ji pi xi bao 24 h,ELISAjie guo xian shi LTAci ji MAP2-3mian yi zu pi xi bao fen bi IL-6,mie huo S.aureusci ji MAP2-3mian yi zu pi xi bao fen bi IL-2、IFN-γhe IL-6。jie lun :mo ni LTAde si fen zhi duo jia kang yuan tai (MAP2-3)neng gou you xiao you dao te yi xing ti ye mian yi ying da he mian yi ji yi ,bao hu BALB/cxiao shu di yu S.aureusgan ran 。zhe yi gong zuo wei S.aureusyi miao yan jiu di gong xin de ce lve 。
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论文作者分别是来自南方医科大学的易夏玉，发表于刊物南方医科大学2019-09-02论文，是一篇关于金黄色葡萄球菌论文,疫苗论文,模拟肽论文,免疫反应论文,免疫记忆论文,南方医科大学2019-09-02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，资料来自南方医科大学2019-09-02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：金黄色葡萄球菌论文; 疫苗论文; 模拟肽论文; 免疫反应论文; 免疫记忆论文; 南方医科大学2019-09-02论文;