噬菌体疗法预防细菌性感染的研究进展及发展方向

由于抗生素的不合理使用,引起了细菌多重耐药性、药物残留、食品安全、环境菌群不平衡等问题,严重威胁人类及动物的健康。因此,寻找新型抗菌药物及治疗方案已成为当前的研究重点。噬菌体是感染细菌、真菌等微生物的病毒,自发现伊始便用于治疗细菌感染。与传统抗生素相比,噬菌体具有分布广、易筛选、高度的宿主专一性、增殖能力强、安全性高、研发成本低等优势。随着多重耐药菌的广泛流行,噬菌体治疗细菌感染重新引起了人们的重视。本文简述了噬菌体的生物学特性、噬菌体疗法在预防细菌感染的应用及噬菌体研究发展方向,以期为噬菌体疗法预防细菌性感染提供参考。     

噬菌体在细菌性疾病诊断和治疗中的应用

从重组噬菌体法、生物扩增法、噬菌体触发的离子级联感应技术及耐药性检测4个方面阐述了噬菌体在病原菌检测中的作用;从细菌分型的角度阐述了噬菌体在病原菌分型中的应用;从活噬菌体、裂解酶、药物载体3个方面陈述了噬菌体在细菌性疾病治疗方法中的研究进展,并分析了噬菌体在应用中的优缺点,展望了今后的研究和应用前景。     

金黄色葡萄球菌噬菌体SP-2裂菌特性分析

为筛选高效裂解金黄色葡萄球菌的专性噬菌体,有效防控养殖场耐药病原菌感染,本研究采集奶牛场粪水样本,利用实验室培养的金黄色葡萄球菌分离裂解性噬菌体,鉴定分析噬菌体的理化特征和生物学特性,测定裂菌效率及裂菌谱;通过模拟污染地面,研究该噬菌体的环境杀菌潜力。结果显示,分离到1株双链DNA噬菌体SP-2,属于短尾噬菌体科(Podoviridae),裂菌谱广,不仅能裂解实验室耐甲氧西林金黄色葡萄球菌(MRSA),而且对食源性及医院临床分离的金黄色葡萄球菌也有很好的裂解作用;在体外杀菌试验中,噬菌体SP-2可在3 h内裂解MRSA菌株PNB25;环境消毒模拟试验结果显示,经噬菌体SP-2喷雾处理后,对平板内细菌的杀菌效率高达90.8%。结论,噬菌体SP-2对金黄色葡萄球菌具有高效、种内广谱的裂解作用,有望成为防控金黄色葡萄球菌感染的一种抗菌制剂及环境消毒剂。     

金黄色葡萄球菌噬菌体SP-2裂菌特性分析

为筛选高效裂解金黄色葡萄球菌的专性噬菌体，有效防控养殖场耐药病原菌感染，本研究采集奶牛场粪水样本，利用实验室培养的金黄色葡萄球菌分离裂解性噬菌体，鉴定分析噬菌体的理化特征和生物学特性，测定裂菌效率及裂菌谱；通过模拟污染地面，研究该噬菌体的环境杀菌潜力。结果显示，分离到1株双链DNA噬菌体SP-2，属于短尾噬菌体科（Podoviridae），裂菌谱广，不仅能裂解实验室耐甲氧西林金黄色葡萄球菌（MRSA），而且对食源性及医院临床分离的金黄色葡萄球菌也有很好的裂解作用；在体外杀菌试验中，噬菌体SP-2可在3 h内裂解MRSA菌株PNB25；环境消毒模拟试验结果显示，经噬菌体SP-2喷雾处理后，对平板内细菌的杀菌效率高达90.8%。结论，噬菌体SP-2对金黄色葡萄球菌具有高效、种内广谱的裂解作用，有望成为防控金黄色葡萄球菌感染的一种抗菌制剂及环境消毒剂。     

副鸡禽杆菌抗原表位的筛选与鉴定

用C型副鸡禽杆菌(Apg)单克隆抗体作为靶标,筛选噬菌体展示随机12肽库,从中获得与之特异性结合的噬菌体克隆,建立了一种筛选副鸡禽杆菌表位的方法.测序结果显示,第3轮筛选后,80%的噬菌体克隆具有氨基酸共同序列Y-P-Q(A)WW,舍有上述共有序列的噬菌体克隆不仅可以与靶抗体特异结合,还可与C型Apg细菌竞争结合靶抗体;将编码YSPHQWWLSGAV的DNA片段插入质粒pFliTrx的多克隆位点,转化E.coli G1826并在鞭毛成功展示;该重组菌能与靶抗体特异结合;用重组菌免疫试验鸡能产生C型Apg细菌的特异性抗体;用C型668株Apg攻毒,免疫接种鸡获得37.5%保护.提示该表位肽用于研制鸡传染性鼻炎疫苗具有广阔的应用前景.     

沙门菌烈性噬菌体的分离鉴定及其生物学特性的研究

以1株肠炎沙门菌(CICC21482)为宿主菌,从湖北省某规模化养猪场的污水中分离得到1株烈性沙门菌噬菌体,命名为SP01,并对其生物学特性进行了初步研究。采用双层平板法筛选并纯化噬菌体,观察噬菌斑的形态;采用透射电镜观察SP01颗粒;酚-氯仿法提取SP01核酸,酶切分析其核酸类型。同时对噬菌体SP01的最佳感染复数(multiplicity of infection,MOI)、一步生长曲线、热稳定性、p H稳定性、噬菌体SP01对细菌的裂解曲线及裂解谱等生物学特性进行了研究。结果,噬菌体SP01噬菌斑形态呈圆形、透亮,边界清晰,无晕环,直径约为1~2 mm;电镜下可见噬菌体SP01有一正多面体立体对称头部,直径约63 nm,含非收缩性尾部,尾长约158 nm,为长尾科噬菌体;提取基因组,酶切鉴定该噬菌体核酸类型为双链DNA;当MOI=0.1时,子代噬菌体滴度较高;噬菌体SP01除对本身宿主菌产生裂解外,还裂解猪霍乱沙门菌和都柏林沙门菌;一步生长曲线试验结果显示,其感染宿主菌的潜伏期约为20 min,裂解期约为60 min,平均裂解量约为120 PFU/cell;噬菌体SP01在20~40℃稳定,在p H=4.0~11.0稳定;对体外培养的特定血清型的沙门菌具有良好的裂菌效果。上述结果表明,噬菌体SP01为1株沙门菌烈性噬菌体,对不同温度、p H的环境均有较强的适应能力且杀菌能力较强,具有开发为抗沙门菌制剂的潜力。     

噬菌体制剂沙立宁对鹅源沙门菌感染的抑菌效果研究

为了探讨噬菌体制剂沙立宁对鹅源沙门菌的抑菌效果,本研究选取血清型分别是鼠伤寒沙门菌和乙型副伤寒沙门菌的2株鹅源沙门菌,首先利用改良寇氏法计算2株菌的半数致死量(LD_(50)),然后利用2株菌人工感染3日龄SPF雏鸡,再用噬菌体制剂沙立宁和头孢噻肟处理感染后的SPF雏鸡,观察雏鸡的临床症状及内脏解剖学变化,检测肝和盲肠的载菌量及两种组织的病理组织学变化。结果显示,鼠伤寒沙门菌和乙型副伤寒沙门菌的LD_(50)分别为1.43×10~7CFU/mL和4.40×10~6CFU/mL;感染2株沙门菌后,雏鸡表现精神萎顿、垂头呆立、闭眼昏睡、绒毛松乱、食欲减退、排白色浆糊状稀便,严重者全身抽搐、呼吸困难,甚至死亡。雏鸡肝中的载菌量随着感染时间延长呈先增加后减少的现象,盲肠载菌量则表现为逐渐减少。组织学方面,肝细胞出现肿胀、胞质疏松、炎性细胞浸润现象,盲肠则表现出固有层炎性细胞浸润、绒毛断裂现象。利用噬菌体制剂沙立宁和头孢噻肟治疗后,雏鸡肝和盲肠载菌量明显减少,两种组织形态趋于恢复正常,炎症反应减轻或消失。结果表明,鹅源沙门菌可以感染雏鸡,噬菌体制剂沙立宁和头孢噻肟对2株鹅源沙门菌的感染均具有抑制效果。     

产志贺毒素大肠杆菌噬菌体vB_EcoM_PHB05的生物学特性及全基因组分析

产志贺毒素大肠杆菌又称产Vero细胞毒素大肠杆菌,是一种可引起水样腹泻、溶血性尿毒综合征、出血性结肠炎等的人兽共患肠道致病菌。以一株产志贺毒素大肠杆菌为宿主菌,从湖北某规模化养猪场的污水中分离得到一株烈性产志贺毒素大肠杆菌噬菌体,命名为v B_Eco M_PHB05。采用双层平板法筛选并纯化得到一株噬菌体,采用透射电镜观察噬菌体颗粒;使用Ion Torrent测序平台进行噬菌体全基因组测序,同时对噬菌体最佳感染复数(MOI)、一步生长曲线、裂解谱等生物学特性进行研究。结果表明,噬菌体v B_Eco M_PHB05在电镜下可见有一正多面体立体对称头部(直径约72 nm),含收缩性尾部(尾长约45 nm,尾宽20 nm),为肌尾科噬菌体;提取基因组,酶切鉴定该噬菌体核酸类型为双链DNA;其最佳感染复数为0.1;一步生长曲线试验结果显示,噬菌体v B_Eco M_PHB05潜伏期约为20 min,平均裂解量约58 PFU/cell;基因测序结果表明,该噬菌体全基因组全长147 659 bp,G+C含量37.5%。噬菌体v B_Eco M_PHB05除对本身宿主菌产志贺毒素大肠杆菌(Escherichia coli O34)产生裂解外,还裂解其它产志贺毒素大肠杆菌和沙门菌。噬菌体v B_Eco M_PHB05为一株广谱烈性噬菌体,其基因组中不含耐药基因及毒力基因,具有开发为抗产志贺毒素大肠杆菌制剂的潜力。     

猪流行性腹泻病毒M蛋白亲和肽的筛选及其抗病毒作用

把成功构建的猪流行性腹泻病毒(PEDV)M蛋白的阳性重组质粒pET-32a(+)-M在大肠杆菌表达系统中进行诱导表达。以表达纯化的M蛋白作为靶蛋白,利用噬菌体展示技术对随机十二肽库进行筛选,4轮淘选后,挑选出了10个与PEDV及M蛋白特异性结合力高的噬菌体单克隆,序列测定后推导的氨基酸序列分别为AGWYCTEVLCV、AYCTRHVCYLDN,多肽被命名为M2、M9。就合成多肽体外抗病毒的效果而言,间接免疫荧光试验和实时荧光定量PCR试验证明,多肽M2、M9及多肽M2与M9联合使用均能够有效抑制病毒复制,且都呈剂量依赖性。其中多肽M2抑制病毒的作用比多肽M9的高,两种多肽联合抗病毒的作用最好。上述结果为进一步研究PEDV M蛋白功能性配体和研制防治PEDV的小分子治疗制剂提供了试验依据,奠定了理论基础。     

小肠结肠炎耶氏菌噬菌体的分离和鉴定

小肠结肠炎耶氏菌(Y.enterocolitica,Y.e)是一种分布很广的人畜共患性疾病病原菌,猪的带菌或发病与该菌在人群流行有密切关系。关于从猪标本中分离出Y.e已屡有报道,我们也曾从屠宰场污水中检出Y.e。鉴于噬菌体与相应细菌在生态学分布上的关联性,我们对猪场污水进行了Y.e噬菌体的分离和鉴定。     

传染性腔上囊病病毒VP3蛋白模拟表位的筛选

为了研究传染性腔上囊病病毒(IBDV)的致病机理及研制有效的IBDV疫苗,利用噬菌体展示技术对IBDV VP3抗原表位进行了筛选。以4株IBDV VP3单克隆抗体HRB-3F、HRB-7B、HRB-7C和HRB-10E作为筛选分子,对噬菌体随机15肽库进行3轮吸附-洗脱-扩增淘洗,从每株单克隆抗体筛选到的噬菌斑中随机挑取20个单克隆噬菌斑,通过间接ELISA和竞争抑制ELISA检测,共选出13个单克隆噬菌斑,经噬菌体gⅢ部分基因的核苷酸序列测定,确定了8个15肽为IBDV抗原表位。这8个15肽在一级结构上没有3个以上连续氨基酸与IBDV Gx(Gen-Bank登录号:AY444873)VP3的氨基酸序列相同,但二级结构上均以β折叠为主,并且与单抗的结合可被VP3蛋白有效地抑制。证实,筛选的是IBDV VP3的模拟表位。     

病毒受体及其研究进展

介绍了病毒受体的基本生物学特性,重点阐述了噬菌体展示、免疫共沉淀及酵母双杂交等病毒受体研究方法,综述了几种重要动物病毒细胞膜受体的研究进展,并对病毒受体研究的发展前景进行了展望。     

日本血吸虫保护性单克隆抗体SSj14识别模拟表位的筛选及其免疫原性分析

为分析日本血吸虫保护性单抗SSj14的模拟表位,观察其免疫保护作用,利用SSj14单抗筛选富集噬菌体的随机六肽库,应用免疫印迹法确定阳性克隆并分析其核苷酸序列,采用竞争ELISA法分析阳性克隆的抗原性,用筛选的阳性噬菌体克隆免疫小鼠进行日本血吸虫病免疫保护试验,计算减虫效果,并用ELISA法检测小鼠免疫前后的特异性抗体水平。结果显示,经3轮富集筛选后,免疫印迹法鉴定到4个阳性克隆,它们对SSj14单抗的抑制率分别为20.0%、40.8%、58.6%、61.5%,并分别在小鼠中诱导了7.42%、9.43%、11.74%、22.30%的减虫率。用噬菌体克隆免疫的小鼠均产生了较高水平的特异性抗体。结果表明,筛选到的阳性噬菌体克隆和SSj14单抗有较高的亲和力,并诱导了抗小鼠日本血吸虫的部分保护效果。     

细菌的CRISPR/cas免疫及免疫识别

综述了40%真细菌以及几乎所有古细菌基因组内存在的CRISPR位点以及细菌的CRISPR/cas免疫机制。主要从CRISPR/cas免疫的抗感染机制、特异性间隔序列的获取、crRNA的成熟以及免疫识别等方面展开论述,阐述了成簇存在的、被短的重复回文序列所分割的非自身基因(CRISPR位点)以及通过类RNA干扰(RNAi)机制特异性地抵抗噬菌体及侵袭性质粒的二次感染。提出此技术在临床上用来抑制抗生素抗性质粒以及毒力因子在病原菌中传播的应用前景。     

鸡白痢沙门菌基因组差异片段的筛选与分析

采用抑制性消减杂交技术(SSH)对鸡白痢沙门菌C79-13株与鸡伤寒沙门菌Sg9株进行了基因组差异片段分析。结果,从C79-13株中共检出13个特异性差异片段。经同源分析,这些序列可分为3类:噬菌体相关序列、质粒相关序列和已知功能序列。这些差异片段包含一些重要的沙门菌毒力相关基因,如编码大肠杆菌素I、paJ蛋白、尾突蛋白、切除酶的基因。结果表明,鸡白痢沙门菌C79-13株与鸡伤寒沙门菌Sg9株基因组间存在较多差异基因,这些差异片段为确定鸡白痢沙门菌特异性遗传标志,建立分子鉴别新体系提供了基础。     

A Conceptual Framework for Modeling Automated Negotiations in Multiagent Systems

In fully automated e‐negotiation all involved parties are software agents, so negotiation takes place in a multiagent system between software agents that have been developed as a computer system for automating tasks in a specific application domain. A multiagent system is a group of agents that interact and cooperate with each other to fulfill their objectives or to improve their performance. How do these agents negotiate with each other to manage their task interdependencies? What negotiation mechanisms are needed? These are important questions. In this article, we present a conceptual framework for modeling and developing automated negotiation systems. This framework represents and specifies all the necessary concepts and entities for developing a negotiation system as well as the relationships among these concepts. This framework can also be used to model human negotiations scenarios for analyzing these types of negotiations and simulating them with multiagent systems. The work reported in this article is the first unified framework that represents all the needed elements for modeling and developing automated negotiation systems and existing relationships between them.     

Intergovernmental Organizations,Interaction, and Member State Interest Convergence

This article presents a general theory of how the interaction of state agents within intergovernmental organizations (IGOs) leads to a convergence in member state interests over time. The theory is based on the notion that, all else being equal, IGOs that facilitate more interaction between individuals from various states are conducive to greater member state interest convergence over time because there are more opportunities for agents from one or more member states to persuade agents from other member states to accept new ideas that affect how they define their states’ interests. I argue that such persuasion does not necessarily have to involve a shift in state identities but can also involve a diffusion of ideas about cause-and-effect relationships. Also, by focusing on IGOs as structures within which state agents interact, I argue against a narrow focus on socialization defined as the induction of new members into community norms. I present three hypotheses regarding which institutional attributes are conducive to member state interest convergence and test them using an original IGO data set. The findings are supportive of my general theory but provide some interesting support for existing theory that runs counter to one of the hypotheses presented here.     

Agents and Structures: Two Views of Preferences, Two Views of Institutions

Two analytical distinct approaches to the study of domestic politics have been referred to as the "new institutionalism." The fundamental difference between the two brands of institutionalism can be seen in the way they handle the relationship between "agents" and "structures.""Structure-based" approaches to institutions give ontological primacy to structures and view agents as being constituted by them. "Agency-centered" approaches view human agents as ontologically primative and view institutions as structures that are created by goal-maximizing individuals. The two approaches are compared, with special attention given to the way they treat the preferences that actors hold. I argue that contrary to arguments made by many structure-based theorists, the agency-centered approach is capable of contributing to discussions regarding the sources of actor preferences. A limited information model of the strategic interaction between workers and capitalists is used to demonstrate ways in which the agency-centered approach can begin to make preferences endogenous.     

猪瘟病毒E2蛋白B细胞识别基序的筛选

根据猪瘟病毒(classical swine fever virus,CSFV)E2蛋白的序列设计并人工合成18条重叠多肽,与载体蛋白BSA偶联后,利用抗CSFV E2蛋白单克隆抗体SWmAb8筛选该蛋白上的B细胞表位;同时用12肽噬菌体随机肽库淘选E2单抗识别多肽序列。结果显示,多肽序列VSPTTLRTEV能被单抗SWmAb8识别;含SPTxLR基序的噬菌体能被单抗SWmAb8结合,且能被病毒抗原竞争性抑制。证实SPTxLR基序很可能是E2蛋白的线性表位之一,可诱导机体产生中和抗体。     

'New learning in old organisations': Children's participation in a school-based nutrition project in western Kenya

The integration of learning into community development processes and how that learning can stimulate positive change pose challenges that development practitioners have met with mixed success. Who the most effective change agents are, how they can be supported, and how their efforts can be diffused in the community and scaled up are key questions in the community development literature. The authors designed and implemented an action-research project in western Kenya on traditional vegetables, recruiting pupils as co-researchers. The purpose of the research was two-fold. One goal was to explore the feasibility of increasing the intake of traditional vegetables through a school-based horticulture programme. The other was to increase pupils' competence as effective change agents by empowering them in culturally compatible ways. The results offer lessons for practitioners regarding creative means to identify and empower change agents within traditional organisations and encourage innovative creation and diffusion of knowledge.