这项工作是由研究经费从医学研究委员会G0400268和MR / K018396 / 1的支持，并通过行动医学研究。进一步支持由国家卫生研究院的研究伦敦大学学院医院的生物医学研究中心提供。

在这项研究中进行的所有动物实验符合国家和欧洲立法和批准了药剂的UCL学院和伦理委员会的英国内政部（HO）。在何项目进行的所有动物上岗证PPL二千二百四十三分之八十〇和PPL七千七百七十三分之七十〇。 1.鼠准备<ol><li>开展所有在体内使用无病原体的Wistar新生大鼠的实验。 </li><li>保留所有的鼠窝（每个群体12例新生儿）在单独的笼子与哺乳期的母亲（9 - 10周龄雌性），在最佳条件（19岁 - 21°C，45 - 55％的湿度，15 - 20换气/小时12小时光照/黑暗周期）。 </li></ol> 2.细菌细胞的制备<ol><li>商店E.大肠杆菌 K1的库存量的20％（体积/体积）甘油在-80℃下。对于每个实验，板材缺货细菌到穆勒，寒春（MH）琼脂平板上，并在37°CO / N。 </li><li>喂奶前，inocu一天迟10毫升MH肉汤与单个大肠杆菌大肠杆菌 K1的殖民地，文化O / N在37℃，200转。作为介质污染的控制，制备10毫升未接种MH肉汤的孵育在37℃下以200rpm。 </li><li>转移100μl的O / N的细菌悬浮液的进9.9 MH肉汤中的溶液（1：100的体积/体积），并孵育在37℃，200rpm下，直到指数中期阶段达到时，对应于0.6的光学密度在600nm（OD 600）的波长进行测定。 </li></ol> 3.喂养新生鼠的E.大肠杆菌 K1 <ol><li>轻轻地从颈背垂直放置在一只手的动物，允许嘴巴得以打开。慢慢插入无菌移液管尖端插入动物的嘴，以及移液管将20μl接种物（〜37℃）到动物的口，在30秒的时间周期。喂奶后立即返回了动物母亲。 </li><li>验证4 - 8×10 6细菌已被送入吨o分别小狗通过连续稀释在PBS中的接种物，并点样到MH琼脂平板上。 </li></ol>由E. 4.评估殖民化大肠杆菌 K1 <ol><li>轻轻地从颈背握住动物在一只手中。浸湿无菌棉签在无菌PBS，然后轻轻擦拭肛周区域。擦拭后立即返回动物的母亲。 </li><li>将拭子末端到含有300微升无菌PBS的管并储存在冰上。 </li><li>通过在PBS中的系列稀释确定的存活细菌的数量和电镀到麦康凯琼脂平板上。 </li><li>确定可行的E.大肠杆菌 K1由噬菌体药敏试验。挑选使用无菌微生物环状单菌落，浸入200微升无菌PBS，然后进行涡旋混合。 </li><li>在一条直线上用无菌微生物环状物，亚培养至MH琼脂上。让板干燥30秒，然后吸取10微升脊梁的teriophage K1E（10 9噬斑形成单位/ ml（PFU / ml）的）上线的中心。孵育板O / N在37℃。 </li><li>第二天，检查板噬菌体介导的裂解。 </li></ol>疾病严重程度的评估5 <ol><li>使用表1中所列的7分制每日5次-评估各动物4的疾病的严重程度。 </li><li>如果动物分数≥3出7，立即宰杀的动物，以减少痛苦。记录动物的死亡。 </li></ol> 6.安乐死新生大鼠和采血车<ol><li>轻轻握住动物在一方面，露出的头部和颈部。用酒精棉签擦拭消毒动物的脖子上。消毒大对使用70％的乙醇剪刀，清洗无菌PBS去除微量乙醇之前。 </li><li>轻轻直接持有的上述动物的皮氏培养皿。杀头使用锋利的surgi新生儿卡尔剪刀，确保血液滴入培养皿。避免与磁头，以防止血液污染皮肤菌群接触。 </li><li>立即使用无菌移液管收集血液，并与肝素的钠盐混合，在20的浓度 - 在0.5 ml离心管50单位/ ml。 </li><li>通过在PBS中的系列稀释确定的存活细菌的数量和电镀到麦康凯琼脂平板上。确定可行的大肠杆菌的数量大肠杆菌 K1通过检测活细菌的敏感性噬菌体K1E。 </li></ol> 7.解剖<ol><li>继新生大鼠断头，用无菌条件下切除和收集感兴趣的组织。洗在70％乙醇和无菌PBS中的所有仪器（ 图1中示出）。 </li><li>清洁夹层板并用70％的乙醇完全润湿腹部。放置在它的后面的尸体和通过（i）固定在解剖板上钉的左后腿来用无菌针的动作表，（ⅱ）拉伸尸体和钉扎的右前肢的动作表，（ⅲ）钉扎的右后腿和左前腿的动作表。 </li><li>拉沿使用镊子下腹部的左手侧的皮肤上，然后沿着从下腹部到使用小解剖剪胸骨尸体的左手侧切断。 </li><li>延伸的切除穿过胸骨，然后向下从胸骨到使用镊子和小解剖剪小腹尸体的右手侧，以确保无下层结构的损坏。 </li><li>最后，利用虹膜弯钳解剖暴露腹膜轻轻拉下皮瓣从胸骨到小腹。 </li><li>轻轻抬起用镊子将腹膜和垂直切开，露出内脏，以确保没有潜在的器官受损。 </li></ol> 8.收集胃肠道<ol><li>识别胃，小肠，盲肠，结肠和肠系膜淋巴系统。 </li><li>轻轻横切在两侧取出胃，再放入含有1.6毫升无菌PBS用无菌镊子一个小巧玲珑。 </li><li>样结肠的直肠。小心地将整个肠道大众使用解剖镊和地点在无菌培养皿中的尸体。确保做到这一点轻轻地使肠道结构和肠系膜淋巴结构不分开。 </li></ol> 9.分离的胃肠道和肠系膜淋巴系统（图2） <ol><li>倾30毫升无菌PBS放入培养皿中，以确保在胃肠道被完全淹没。 </li><li>确定肠系膜淋巴系统的核心质量，并掐使用精细解剖钳。找出小肠最近端和捏用细镊子剥离。 </li><li>慢慢地将CENTRA肠系膜淋巴系统和远端小肠中沿相反的方向，直到两个组织的升质量都完全彼此分离。小心执行此过程，以确保小肠未拉伸的，因为这可以防止近侧，中间和远侧区的重现性集合。将肠系膜淋巴系统中的300 - 500微升无菌PBS，并放置在冰上。 </li></ol> 10.切片胃肠道<ol><li>横切GI道在盲肠从结肠分离小肠。 </li><li>将在300结肠 - 500微升无菌PBS，并置于冰上。 </li><li>收集来自小肠的近端，中间和远端区域的代表性组织。对齐从中点小肠。 </li><li>然后，（ⅰ）收集的最后2厘米之前盲肠作为远端小肠组织的，（ⅱ）收集由5所述组织 - 6厘米以上的中点作为近端小肠，并（ⅲ）收集来自3所述组织 - 下方5厘米的中点作为中间小肠。 </li></ol> 11.收集肝脏<ol><li>切除的胃之后识别大鼠肝脏的三波瓣内。 </li><li>叶拉离用细镊子解剖腹腔。 </li><li>切割用细剪刀任何附加韧带取出肝脏。将肝脏在300 - 500微升无菌PBS，并置于冰上。 </li></ol> 12.收集脾<ol><li>确定脾脏。 </li><li>脾拉离胃和用细剪刀剪开胃脾韧带。 </li><li>将脾脏在300 - 500微升无菌PBS，并置于冰上。 </li></ol> 13.收集肾脏<ol><li>鉴定肾脏，将在腹腔中取出时对胃肠道和其他器官的背面变得可见。 </li><li>切输尿管及任何附韧带ü唱精细解剖剪刀，用细镊子取出肾脏。删除肾上腺和用细镊子和剪刀剥离任何脂肪物质（如果还连着）。 </li><li>将两个肾脏在300-500微升无菌PBS，并放置在冰上。 </li></ol> 14.收集脑<ol><li>断头后，按住头用弯钳垂直位置。掐皮肤上的头部纵向使用另一套弯钳的顶部，并用细解剖剪一个切口。切颈部附近剩余的皮肤，再纵向与精细解剖剪刀。 </li><li>采用两侧细镊子揭示头骨和删除使用精细解剖剪刀皮瓣拉扯皮肤向外的方向。将精细解剖剪刀脊柱开幕，并沿走向主席台颅骨切开。 </li><li>用细镊子的方向向外拉头骨的两个部分。切去除颅骨段。 </li><l我&gt;广泛使用镊子，使用从朝主席台脊柱开幕向上运动舀取出大脑。洗了脑在PBS两次，以去除任何血液斩首的过程。 </li></ol> 15.组织处理和均质化<ol><li>对于需要生存能力计数或DNA提取，放置组织成含有无菌PBS试管实验。立即通过在PBS中系列稀释，并镀上麦康凯琼脂平板上，或存储在-20℃下用20％的甘油用于短期测定存活菌数。对于DNA的提取，存储组织于-20℃下。 </li><li>对于需要的RNA提取，放置组织成的RNAlater溶液适当体积（每1毫克组织10微升）的实验中，然后立即储存在-20℃下短期或在-80℃下长期贮存。 </li><li>对于实验需要成像，地方的组织在10ml Methacarn溶液（60％甲醇，30％的氯仿和10％乙酸），那么存储吨室温（20 - 25℃）达3周。 </li><li>按重量计管的前和加入组织后的比较计算组织重量。均质使用匀浆组织。在无菌PBS中的各样品的均质化之间洗一次均化器在70％乙醇和两次。 注：Methacarn固定是期望的肠样品的固定，以保持粘蛋白相关的粘膜防御结构;福尔马林固定可用于全身组织。 </li></ol>

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B., Luzio, J. P., Taylor, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+and+cure+of+systemic+Escherichia+coli+K1+infection+by+modification+of+the+bacterial+phenotype.">Prevention and cure of systemic Escherichia coli K1 infection by modification of the bacterial phenotype.</a> Antimicrob. Agents Chemother. 48 (1), 1503-1508 (2004).</li><li>Achtman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six+widespread+bacterial+clones+among+Escherichia+coli+K1+isolates.">Six widespread bacterial clones among Escherichia coli K1 isolates.</a> Infect. Immun. 39 (1), 315-335 (1983).</li><li>Mushtaq, N., Redpath, M. B., Luzio, J. P., Taylor, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+experimental+Escherichia+coli.+infection+with+recombinant+bacteriophage-derived+capsule+depolymerase.">Treatment of experimental Escherichia coli. infection with recombinant bacteriophage-derived capsule depolymerase.</a> J. Antimicrob. Chemother. 56 (5), 160-165 (2005).</li><li>Gross, R. J., Cheasty, T., Rowe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+bacteriophages+specific+for+the+K1+polysaccharide+antigen+of+Escherichia+coli.">Isolation of bacteriophages specific for the K1 polysaccharide antigen of Escherichia coli.</a> J. Clin. Microbiol. 6 (6), 548-550 (1977).</li></ol>

作者宣称，他们有没有竞争的财务权益。

此处所描述的动物模型基础上，任何旨在再现天然存在的感染在人中的显着特征以前的工作。新生大鼠最初用于研究由于H.婴儿脑膜炎b型流感的品种满足主要标准感染稳健的模型。因此，相关病原体的进入门应该反映人类的自然感染的和可重复给予上升到足够的时间相似病理以允许治疗性干预。该技术中使用的，不应限制本过程的适用性，并且不应向疾病结果20。 H的模型性脑膜炎由莫克森和他的同事开发出幼鼠满足这些标准的20;上呼吸道和这一重要特征的粘膜的定植用非创伤复制在幼鼠后发生的自然感染细菌的抽动滴注到鼻腔的膜。重要的是，感染的年龄依赖性的性质被复制在模型中。 同组也是率先开发E的一种非侵入性的模型大肠杆菌 K1 NBM在新生大鼠22。无病原体的Sprague-Dawley幼鼠，通过口服胃管喂食10月8日至10月10 日的细菌定植;因此接种物比由我们采用的相当高的。殖与三个K1株检查，C94（O7：K1：H-）EC3（O1：K1：H-）和LH（Ø75为：K1：H 3），发生在一个相对高的比例（48-74％） K1喂养的动物，但菌血症，脑膜炎和死亡的发生率是可变的，比定植率显著降低。 大肠杆菌的克隆性质大肠杆菌 K1的实验性感染以后被建立的23和现在是明显的，只有O18：K1和在较小程度上，O7：K1的血清型能始终如一地引起全身感染。出于这个原因，neuropathogenic 大肠杆菌的发病机制的这些调查大肠杆菌 K1是基于使用毒力增强的O18：K1株A192PP。 E.的比较通过胃管新生大鼠，如采用由Achtman的组23为用于使用前一方法，几乎可以肯定，由于损坏的死亡由Glode和同事22，和一个液滴输送方法揭示的数目过多粘膜的表面的大肠杆菌 K1馈送胃管。作为定植率与这两种方法可比较的，则建议使用供给使用移液管，用无菌尖端的细菌的较少侵入性方法，如在此所描述的通信。 在我们的研究中使用的大肠杆菌菌株A192PP是O18：K1。它是临床菌株大肠杆菌的毒性更强衍生物杆菌 A192最初回收从病人败血症27 </SUP&gt;。通过连续传代通过新生大鼠26得到的菌株的毒力增强。应变诱发年龄相关疾病的严重程度，采用100％的菌血症和死亡率当给药至2天大的动物28。与此相反，第9天龄的动物对疾病的完全抗性。 K1的特异性裂解的噬菌体可以用于区分E。大肠杆菌 K1其他E.大肠杆菌菌株29。在这项研究中，存活的细菌的敏感性噬菌体K1E应当用于（ⅰ）检查E的纯度大肠杆菌 K1的混悬剂制备要被馈送到所述动物，和（ii）区分大肠杆菌从其他大肠菌群大肠杆菌 K1，以便计算在肛周拭子，血液和组织样品的可行性。如果菌落E.大肠杆菌 K1，这将是容易的噬菌体K1E裂解，和细菌的生长会在噬菌体接种部位被抑制。如果菌落不是E.大肠杆菌 K1，将BË耐KIE噬菌体裂解，并且应该有细菌生长的噬菌体接种部位的区域。它应该牢记的是，动物模型不能反映自然发生的疾病的所有功能。当前模型可以被修改来检查neuropathogenic细菌比E.其它的毒力特征大肠杆菌 A192PP和变型中的定植接种物的大小可被容纳。该技术的未来应用可能包括急需的药品评价治疗的条件，并揭示宿主对殖民和组织侵犯的细节。 这里所描述的方法是简单而有效的。 10个单胎 - 12幼崽被用作试验组和对照组，这在-垃圾的方法确保重复性和统计有效性的高度。当务之急是对幼仔的任何procedur后尽快回到他们的自然母亲e和窝不应该因此包括动物进行不同的干预措施。重要的是，所供给的接种物被温热否则幼仔将拒绝提供培养。幼仔快速开发复杂的微生物区系，并在出生后两天胃肠道是定植从确定为在婴儿和成人消化道中最丰富的微生物的门类广泛的细菌。还没有被馈送大肠杆菌幼仔大肠杆菌 A192PP不携带E.大肠杆菌 K1在胃肠道17等确定的定植率相对来说比较简单。然而，NEUS的基于定量PCR的检测方法，定植E的大肠杆菌 K1更为敏感，传统的培养方法，并强烈推荐17。

全身性细菌感染是一个主要的威胁福祉和新生儿的存活;早产儿是特别脆弱的。新生儿细菌性脑膜炎（NBM），常与细菌性败血症，仍然是发病率和死亡率的一个显著源在最初的几个星期的生活，问题是阻力的不断发展，以一线抗菌药物1,2加剧。 NBM的情况下，是一种医疗紧急情况具有很高的医疗，社会和经济负担3;因此，目前迫切需要新的治疗剂，特别是新的预防策略，以减少感染的负担。 NBM的一些特征是不寻常的：在发达国家， 大肠杆菌和B组链球菌是负责对大多数情况下与这些菌株引发的NBM的容量几乎总是用保护多糖加利福尼亚州的存在相关联psule，使病原体逃避免疫识别处理4。 neuroinvasive E的- （85％80）的比例非常高大肠杆菌表达的K1的胶囊5,6，α-2,8连接的多聚羧酸系聚合物是结构相同的主机神经元可塑性7的调节剂。 新疗法和避孕药的NBM和相关的菌血症和败血症的评价将明显受益感染强大的动物模型，模拟疾病在人类新生儿的主要特征，尤其是强烈的年龄依赖性和感染的自然途径。广泛用于革兰氏阳性和革兰氏阴性细菌性脑膜炎型号可供选择8,9和这些都大大扩大了我们在这些感染的发病机制，病理生理学和治疗方案的知识。因此，实验性感染的大鼠，小鼠，兔和猴已被用于研究脑膜炎在两个n个eonate和成年人。然而，许多这些车型采用直接脑池内或皮下注射细菌感染引发的，创建一个人造的发病机制绕过传播的自然过程，从定植的网站。在某些情况下，接种的这些方法导致了在病理显著变化;例如， 大肠杆菌的皮下给药大肠杆菌 K1株废止与自然感染相关的年龄依赖性，产生菌血症和侵入中枢神经系统（CNS）中的两个新生儿和成人10。倾向E.大肠杆菌 NBM关键取决于病原体的垂直传播，从母亲到婴儿出生时或出生后11不久。母系来源E.大肠杆菌 K1的细菌定殖于新生儿胃肠（GI）道11-13，这是无菌的，出生但迅速获得一个复杂的微生物区14。在殖民新生儿，E.大肠杆菌K1的细菌有从肠腔穿过血-脑或血-脑脊液屏障9,15进入中枢神经系统之前易位进入全身循环的能力。实验感染的强劲机型的设计应考虑这些细节的考虑。 虽然小鼠中已广泛用于某些形式的细菌性脑膜炎8人的研究，它们不适合于新生儿感染的研究：它们是由全身性感染淹没并没有显示出人类婴儿16的强年龄依赖性特性。此外，α防御素，胃肠道由E.提供保护，防止系统入侵的肽键大肠杆菌 K 1 17，高表达于帕内特细胞和嗜中性粒细胞在人类和大鼠，但不是在小鼠18。有一个在其他的没有发现显着程度的​​重复，冗余性和异质性在小鼠防御及相关cryptidin基因imals 19。新生大鼠最初使用莫克森和他的同事20鼻内接种，调查流感嗜血杆菌性脑膜炎的发病机理，复制这个新生儿病原菌定植的自然景观在人，随后改编为年龄依赖性E.大肠杆菌 K1菌血症和脑膜炎。 Bortolussi 等人 21就业腹腔注射的细菌的接种物以引发感染但Glode的关键研究和同事22使用的口服胃饲胃肠道定植后平行感染的天然途径。作为胃管可能损伤粘膜表面，该过程被进一步完善，该接种物馈送到新生儿23。这里，该方法用于胃肠道定植和用于跟踪描述在易感大鼠幼仔的感染程序;另外，该模型的治疗性和预防性应用进行了讨论。

<table border="0" cellpadding="0" cellspacing="0" width="1042">
	<tbody>
		<tr>
			<td>Pathogen-free Wistar rats (12 x neonate, 1 x lactating mother)</td>
			<td>Harlan, UK</td>
			<td>www.harlan.com</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli K1 A192PP</td>
			<td>Taylor lab</td>
			<td>www.ucl.ac.uk</td>
			<td>Mushtaq et al. 2004</td>
		</tr>
		<tr>
			<td>Bacteriophage K1E</td>
			<td>Taylor lab</td>
			<td>www.ucl.ac.uk</td>
			<td>Mushtaq et al. 2004</td>
		</tr>
		<tr>
			<td>Glycerol&#160;</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>G5516</td>
		</tr>
		<tr>
			<td>Mueller-Hinton Agar</td>
			<td>Oxoid, UK</td>
			<td>www.oxoid.com</td>
			<td>CM0337</td>
		</tr>
		<tr>
			<td>Mueller-Hinton Broth</td>
			<td>Oxoid, UK</td>
			<td>www.oxoid.com</td>
			<td>CM0405</td>
		</tr>
		<tr>
			<td>MacConkey Agar</td>
			<td>Oxoid, UK</td>
			<td>www.oxoid.com</td>
			<td>CM0007</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>P4417</td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>E7023</td>
		</tr>
		<tr>
			<td>Heparin Sodium Salt</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>84020 
			Prepare 20-50 units/ml</td>
		</tr>
		<tr>
			<td>RNAlater Solution</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>R0901 
			10&#181;l/mg tissue</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>320099</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>C2432</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>322415</td>
		</tr>
		<tr>
			<td>Cotton-tipped swabs</td>
			<td>Fisher Scientific, UK</td>
			<td>www.fisher.co.uk</td>
			<td>11542483</td>
		</tr>
		<tr>
			<td>Alcotip Swabs</td>
			<td>Scientific Laboratory Supplies, UK</td>
			<td>www.scientificlabs.co.uk</td>
			<td>SWA1000</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Sigma, UK</td>
			<td>www.sigmaaldrich.com</td>
			<td>P5856</td>
		</tr>
		<tr>
			<td>30mL Universal Tube</td>
			<td>AlphaLaboratories, UK</td>
			<td>www.alphalabs.co.uk</td>
			<td>CW3890</td>
		</tr>
		<tr>
			<td>0.5ml microcentrifuge tubes</td>
			<td>StarLab, UK</td>
			<td>www.starlab.co.uk</td>
			<td>I1405-1500</td>
		</tr>
		<tr>
			<td>1.5ml microcentrifuge tubes</td>
			<td>StarLab, UK</td>
			<td>www.starlab.co.uk</td>
			<td>I1415-1000</td>
		</tr>
		<tr>
			<td>0.1&#181;l calibrated loops</td>
			<td>StarLab, UK</td>
			<td>www.starlab.co.uk</td>
			<td>E1412-0112</td>
		</tr>
		<tr>
			<td>L-shaped spreaders</td>
			<td>StarLab, UK</td>
			<td>www.starlab.co.uk</td>
			<td>E1412-1005</td>
		</tr>
		<tr>
			<td>Cuvettes</td>
			<td>Fisher Scientific, UK</td>
			<td>www.fisher.co.uk</td>
			<td>10594175</td>
		</tr>
		<tr>
			<td>Forceps straight with fine points</td>
			<td>Fisher Scientific, UK</td>
			<td>www.fisher.co.uk</td>
			<td>12780036</td>
		</tr>
		<tr>
			<td>Forceps straight with blunt tips</td>
			<td>Fisher Scientific, UK</td>
			<td>www.fisher.co.uk</td>
			<td>12391369</td>
		</tr>
		<tr>
			<td>Forceps watchmaker&#39;s curved with very fine points&#160;</td>
			<td>Fisher Scientific, UK</td>
			<td>www.fisher.co.uk</td>
			<td>12740926</td>
		</tr>
		<tr>
			<td>Scissors straight with very fine points</td>
			<td>Fisher Scientific, UK</td>
			<td>www.fisher.co.uk</td>
			<td>12972055</td>
		</tr>
		<tr>
			<td>Laboratory Scissors</td>
			<td>VWR, UK</td>
			<td>www.vwr.com</td>
			<td>USBE4251</td>
		</tr>
		<tr>
			<td>25g Syringe Needles</td>
			<td>Greiner Bio-One Ltd</td>
			<td>www.greinerbioone.com</td>
			<td>N2525</td>
		</tr>
		<tr>
			<td>LAMBDA 25 UV/Vis Spectrophotometers</td>
			<td>PerkinElmer, UK</td>
			<td>www.perkinelmer.co.uk</td>
			<td>L60000BB</td>
		</tr>
		<tr>
			<td>Unitemp Incubator</td>
			<td>B&#38;T, UK</td>
			<td></td>
			<td>OP958</td>
		</tr>
		<tr>
			<td>Multitron shaking incubator</td>
			<td>INFORS HT, UK</td>
			<td>www.infors-ht.com</td>
			<td>AJ118</td>
		</tr>
		<tr>
			<td>Ultra-Turrax T-10 homogenizer</td>
			<td>IKA Werke</td>
			<td>www.ika.com</td>
			<td>0003737000</td>
		</tr>
	</tbody>
</table>

non-invasive model of neuropathogenic escherichia coli infection in the neonatal rat

动物宿主和细菌病原体之间的相互作用的研究是唯一有意义的，如果所用的感染模型中复制了自然感染的主要特征。本协议描述由于在新生大鼠neuropathogenic 大肠杆菌 K1全身感染的建立和评价程序。胃肠道的定植导致传播沿感染的肠淋巴血 - 脑当然病原体和模型显示强烈的年龄依赖性。 大肠杆菌的菌株大肠杆菌 O18：K1具有增强毒力的乳鼠产生极高的速度定植，易位血室和脑膜浸润下，通过脉络丛中转。如在人类宿主，中枢神经系统的渗透是伴随着局部炎症和一个总是致命的结果。该模型是经过验证的效用的机制研究发病机制，治疗性干预措施的评价和细菌毒力的评估。

该E.此处描述的大肠杆菌 K1的全 ​​身感染模型复制许多在人体中的自然感染的特征。细菌被摄入，定殖于胃肠道，建立器官特异性疾病与脑24的相关的炎症之前经由肠系膜淋巴结移位到血液舱。重要的是，该模型会显示较强的年龄依赖性;如示于图3中 ，2日龄（P2）的幼鼠极易受到侵入性疾病，但是在一个7天期间，动物变得越来越难熔感染，但不向GI道定植17。从胃肠道定植到血液舱的部位转运后，细菌可以在脉络丛25主要进入中枢神经系统之前被可视化的血液样品中的荧光显微术（ 图4）。在某些动物中，有大量的入侵等主要脏器如肺，脾，肾25。 细菌数目在组织中可以个别幼仔25但之间有很大差异时的生物负载的得分为存在或不存在是有高度再现性的关于机关的入侵。随着12幼崽作为一个单一的测试队列一窝，使用G *电源软件功率计算确定该样本的大小等同于寻找基于生存的使用从队列六只动物和&gt; 99％的概率，如果所有的效果了98.6％的概率12顷考虑。模型，因此适用于专为新生儿细菌性感染的治疗量身新试剂的评价，并已用于在程序中来评价解聚酶EndoE的胶囊，其选择性地移除从细菌表面24-26的K1胶囊的治疗潜力。它也可用于研究宿主细菌相互作用的轻拍该冲击E的hogenesis 大肠杆菌 neuropathogens;在这方面，已经采用了E的研究大肠杆菌 A192PP殖民化和传播。业已证明， 大肠杆菌大肠杆菌 A192PP细胞坚持的P2，P5和P9幼仔大量胃肠道;定植在这三组的时间方面是非常相似的（ 图5），并反映了细菌的复制和维持肠道内人口密度的能力。 将临床分离株A192的毒力是由新生大鼠连续传代，以确保动物使用很少或没有冗余增强。E.大肠杆菌 A192殖民P2新生鼠100％的效率，在动物的35％，引起菌血症和生产的25％27产生致命的影响。在传代衍生A192PP定居于胃肠道，产生菌血症，引起致命性在所有的P2幼崽。因此，该模型可用于研究二的毒力fferent K1菌株相对于它们从定植部位侵入中枢神经系统和其他器官系统的容量。在这种情况下，Pluschke和同事23中使用的乳鼠感染模型来确定的95 大肠杆菌的能力大肠杆菌 K1株人源性引起肠道定植后菌血症;他们观察到的广泛的变化既殖和侵入的能力的效率，支撑大肠杆菌的克隆性质大肠杆菌 K1 neuropathogens。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/52018/52018fig1highres.jpg" width="600" /> 图1.材料组织收集：（A）秤，（B）包含必要的媒体预称重管，（C）手术台上，统治者和针头牵制动物，（D）70％（V / V）乙醇和PBS进行消毒的组织采集设备， <s仲&gt;（E）的组织采集用试剂盒，包括一个大剪刀为断头和剪子和镊子和各种尺寸和形状，（F）中的冰刀片，以保持组织中，（G），70％（体积/体积）的乙醇灭菌在手术台上，并包围。 <a href="https://www.jove.com/files/ftp_upload/52018/52018fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/52018/52018fig2highres.jpg" width="600" /> 图2.分离胃肠道及肠系膜淋巴系统。 （A）的把手的肠系膜淋巴系统以细解剖镊的中央质量。（B）的把手小肠的近端部分，并且拉方向相反。（℃）的胃肠道和肠系膜淋巴系统将完全分隔符即<a href="https://www.jove.com/files/ftp_upload/52018/52018fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/52018/52018fig3highres.jpg" width="500" /> 图3.生存以下E.口服年龄介乎2天（P2）九天（P9）新生幼鼠大肠杆菌 A192PP，说明全身性感染的壮龄相关性，每个组代表24例新生儿。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/52018/52018fig4highres.jpg" width="600" /> 大肠杆菌的图4.荧光图像大肠杆菌 A192PP细胞从P2的小狗血涂片感染细菌之后口服。在细菌表面的脂多糖抗原Ø是STained用兔抗O18多克隆抗体和Alexa546标记的羊抗兔第二抗体。在K1胶囊可视化与EndoE-GFP试剂。事实上，血液样本中检测到的所有细菌所显示的保护K1胶囊。图片由安德烈Zelmer博士抓获。 <a href="https://www.jove.com/files/ftp_upload/52018/52018fig4highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/52018/52018fig5highres.jpg" width="500" /> 图5. E.细菌接种。DNA的大肠杆菌 A192PP肠道定植以下管理是从整个肠道大肠杆菌中提取大肠杆菌 K1的菌落形成单位/ g（菌落形成单位/克）通过定量聚合酶链反应（qPCR的）来确定针对polysialyltransferase（NEUS）基因组织如别处所述17 </suP&gt;。 LOD：检测限。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 特征 </td><td> 健康 </td><td> 不良 </td></tr><tr><td>皮肤的颜色</td><td>粉红色</td><td>淡/黄</td></tr><tr><td>敏捷（翻正反射） </td><td>小狗立刻就扭转落后的位置</td><td>难以扭转落后的位置（&gt; 3秒），或者不能达到</td></tr><tr><td>上腹部轻微的压力</td><td>没有声音</td><td>声音激荡</td></tr><tr><td>胃/乳线</td><td>可见，白</td><td>不可见</td></tr><tr><td>温度</td><td>温暖</td><td>比较冷* </td></tr><tr><td>重量</td><td>每天1.5-2克增益</td><td>没有增重或减重</td></tr><tr><td>当放置在笼子里的行为</td><td>对母亲的动作，并开始˚Feeding </td><td>不能走向母亲，并显示喂养困难</td></tr></tbody></table> 表1. 7分的评分系统：列出的第3得分通常观测到的初步迹象。 *新生儿全身性感染的经验体温升高（&gt; 2℃）。然而，由于缺乏对动物的敏捷性，以达到其母亲以维持体温，不健康的动物可能变得垫料分离和感觉冷到裸手。

Watch this Scientific Journal Video about Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat at JoVE.com

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

这里，一个程序被描述为建立系统性感染的乳鼠大肠杆菌 K1的培养物。这种非侵入性的过程允许定植在胃肠道的，病原体向全身循环的移位，以及侵袭中枢神经系统的脉络丛。

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

non-invasive-model-neuropathogenic-escherichia-coli-infection

Research

JoVE Journal

Immunology and Infection

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

10.8K 次观看.  University College London. 这里，一个程序被描述为建立系统性感染的乳鼠大肠杆菌 K1的培养物。这种非侵入性的过程允许定植在胃肠道的，病原体向全身循环的移位，以及侵袭中枢神经系统的脉络丛。 

视频: Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

科研

免疫与感染

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

播散性念珠菌在斑马鱼幼虫的非侵入性成像

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

非侵入性光学成像淋巴血管的鼠标

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

一步法负色谱纯化<em&gt;幽门螺杆菌</em&gt;中性粒细胞激活蛋白过度表达<em&gt;大肠杆菌</em&gt;在批处理模式

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

在大肠杆菌中生成特异的乙酰蛋白的简易协议

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...