我们感谢 Dr. R 斯托克斯为波士顿巴斯德菌株和 a. 技术支持。我们还感谢金瑞对基因合成的帮助。

所有动物都按照不列颠哥伦比亚大学动物保育和使用委员会批准的协议进行维护。实验得到动物保育和使用委员会的批准, 并按照加拿大动物保育委员会的指导方针进行。动物保证福利号是 A11-0247。
1. 表达质粒的单体素融合蛋白的生成
<ol><li>子克隆单体素序列12成 pDEST17 质粒之间的 "CTC" 和 "GAA" 网站对应的 133-134bp. (即, 介于 6-histag 和 pDEST17 Attr1重组站点) 之间以获得 p17-Avi。 注: 单体素 DNA 序列如下图所示。三突变引入野生型素获得单体素显示在粗体字符。 gccagaaagtgctcg ctgactggga aatggaccaa cgatctgggcATCatga ccatcggggc tgtgaacagc agaggtgaat tcacaggcac ctacatcaca gccgtaacag ccacatcaaa tgagatcaaa gagtcaccac tgcatgggac acaaGCTacc atcaacaaga ggacccagcc cacctttggc ttcaccgtca attggaagtt ttcagagtcc accactgtct tcacgggcca gtgcttcata gacaggaatg ggaaggaggt cctgaagacc atgtggctgc tgcggtcaag tgttaatgac attggtgatg acAAAaaagc taccagggtc ggcatcaaca tcttcactcg cctgcgcaca cagaaggagt</li>
	<li>设计底漆两侧的 B1 和反鱼雷B2 网站对应的卵子多肽757-1035 (I-A b-和 H-2Kb限制表位) DNA 序列。底漆序列是: b1-OVA GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCTTGAGCAGCTTGAGAGTAT 和卷 b2-OVA GGGGACCACTTTGTACAAGAAAGCTGGGTGTTACCCTACCACCTCTCTGC。(b1 和 b2 序列是粗体的. <ol>
<li>PCR 放大卵子多肽757-1035 DNA 序列与上面的引物 pUC57-OVA 质粒作为模板。</li>
		<li>通过特定于站点的体外重组反应 (如, BP Clonase) 将 PCR 产物克隆成 pDONR-221, 以获得 pDONR 卵。</li>
	</ol>
</li>
	<li>利用 LR Clonase 反应将感兴趣的基因转移到 p17-Avi, 获得 p17-Avi-OVA。 注: 有关 BP 和 LR 重组克隆的详细信息, 请参阅制造商手册。</li>
</ol>2. 单体素融合蛋白的表达、纯化和复用
<ol><li>将 p17-Avi-OVA 质粒转化为大肠杆菌 BL21 并诱导250毫升的大肠杆菌 BL21 培养与 IPTG (0.1 米) 3 小时在37° c. </li>
	<li>
细菌和包涵体的溶解
	<ol>
<li>250毫升的诱导 BL21 培养30分钟离心在 4000 x g 和4° c。</li>
		<li>收集10毫升的裂解缓冲液 (50 毫米三盐酸, 150 毫米氯化钠, 6 米胍-hcl, pH 1.5-2.5) 和孵育15分钟在95° c。在一个旋转的室温下通宵孵育。</li>
		<li>离心机30分钟在 1.5万 x g 和4° c 和收集上清液。</li>
	</ol>
</li>
	<li>
用镍 NTA 柱纯化上清 (溶解包涵体分数) 的 Avi 卵。
	<ol>
<li>用裂解缓冲液稀释包涵体1:2。</li>
		<li>每250毫升的文化衍生包涵体使用4毫升的镍 NTA 树脂。</li>
		<li>用10毫升的溶解缓冲液 (pH 7.0) 冲洗3x。</li>
		<li>洗与10毫升的洗脱缓冲液 (裂解缓冲液 pH 7.0 与250毫米咪唑)。</li>
	</ol>
</li>
	<li>再洗蛋白由渐进稀释 (1:10) 和快速涡流在三缓冲 (pH 7.5) 含有1毫米的双制式, 200 毫米 NDSB256 (3 (Benzyldimethylammonio) 磺), 0.5 毫米吐温 80, 500 毫米精氨酸, 200 毫米氯化钠为30分钟室温。</li>
	<li>De 盐和缓冲交换折叠蛋白质从三缓冲入 PBS 与蛋白质集中器根据制造商的协议。</li>
	<li>在 3500 x g和4° c 的情况下, 用离心法去除聚合体30分钟, 并在-20 ° c 储存可溶性蛋白等分。</li>
</ol>3. BCG 细胞表面法
<ol><li>种植卡介苗在 Middlebrook 7H9 汤与 10% OADC (油酸, 白蛋白和葡萄糖溶液) 和0.05% 吐温80在37° c 在一个振动筛平台在 50 rpm 直到 OD = 0.5-1。 注: bcg 是生物安全等级2病原体, 所有有关 bcg 的实验都应在生物安全柜内进行, 并配备适当的个人防护设备。</li>
	<li>洗涤 109 BCG 3 次与500µL 的冰冷内毒素免费 PBS 加 0.1% Tween80 (PBST) (pH 8.0)。在无菌内毒素的 PBS 中悬浮卡介苗颗粒。</li>
	<li>
立即使用前, 准备1毫升的10毫米磺-NHS SS 生物素在无菌过滤水中。
	<ol>
<li>孵育细菌与1毫升的0.5 毫米磺-NHS SS 生物素在室温下为30分钟。</li>
	</ol>
</li>
	<li>
用500µL 的冰冷 PBST 清洗标记的细菌3次, 以去除非反应的法试剂。
	<ol>
<li>再悬浮颗粒在1毫升的 PBST。</li>
	</ol>
</li>
	<li>
为了评估法效率, 染色 108化卡介苗与亲和 FITC (1:100, 25 µL) 在室温下20分钟。
	<ol>
<li>在室温下孵育1毫升2% 粉煤灰中的染色菌, 然后通过流式细胞仪分析对法的含量进行检测。</li>
	</ol>
</li>
</ol>4. 化分枝杆菌的表型: 生长和存活
<ol><li>
用 10% OADC (油酸、白蛋白和葡萄糖溶液) 和0.05% 吐温80在37° c 的 Middlebrook 7H9 汤中生长 BCG 菌株, 在 50 rpm 的振动筛平台上。
	<ol>
<li>在8天的时间内记录细菌培养的光学密度 (OD600)。</li>
	</ol>
</li>
	<li>
使用 BCG-卢克 (以前描述的9) 来检测巨噬细胞中细菌的存活。
	<ol>
<li>感染 RAW264.7 细胞与化卡介苗-卢克 (莫伊 10:1) 或未经治疗的 bcg-卢克 (控制) 和孵化在37° c 超过48小时的时间内。</li>
		<li>冲洗细胞单分子, 然后溶解 0.025% SDS 释放摄入的细菌。</li>
		<li>用荧光检测系统和计测量生物发光的生产。 注意: 发光信号是细菌生存能力的指示。有关荧光化验的详情, 请参阅制造商手册。</li>
	</ol>
</li>
</ol>5. 单体素融合蛋白与化卡介苗表面的结合
<ol><li>混合 5 x 108化卡介苗与 Avi 蛋白 (10 微克/毫升决赛在 PBS t) 为 1 h 在室温下在振动筛平台。</li>
	<li>洗涤细菌3次与500µL 冰冷 PBST 和着色兔抗素抗体 (Ab) (1:100 稀释, 先前描述的10) 为20分钟室温, 然后与 FITC 共轭山羊抗兔 IgG Ab 在相同的条件。</li>
	<li>用500µL PBST 洗涤细菌3次, 用流式细胞仪分析表面装饰的程度。</li>
</ol>6. 分枝杆菌冻干
<ol><li>Biotinylate 细菌根据步骤 3.1-3.4 和外套化细菌与 Avi 卵根据步5.1。</li>
	<li>分化和涂布细菌 (108), 用 PBS 洗涤, 再在0.5 毫升冻干培养基中重新悬浮 (25% Sauton 培养基、75% H2O 和 1.5% Na 谷氨酸)。</li>
	<li>将细菌转移到玻璃瓶中, 在一夜之间冷冻-80 ° c 冷冻。</li>
	<li>Lyophilize 用冷冻干燥机为24小时装满和冰冻的玻璃瓶。</li>
	<li>在室温下储存干燥样品, 必要时在 PBS 中进行重组。</li>
	<li>重复步骤3.5 以评估法和表面涂层的稳定性。</li>
</ol>7. 吞噬试验
<ol><li>在10厘米直径培养皿中生长生264.7 巨噬细胞, 在含有 5% FCS 的 DMEM 培养基中, 1% 每种 l-谷氨酰胺、HEPES、非必需氨基酸、青霉素和链霉素直到 70-80% 70-100。</li>
	<li>种子 2 x 106生264.7 巨噬细胞在 6-井板中。允许单元格在37° c 和 5% CO2的夜间粘附。</li>
	<li>根据步骤 3.1-3.4 和 5.1, 生成 DsRed bcg (以前描述的9), 用 avi 卵 (DsRed 卡介苗-avi-卵) 装饰。</li>
	<li>用 DsRed 卡介苗-Avi-卵或未经处理的 DsRed 卡介苗感染原细胞, 在莫伊20:1 为24小时, 在37° c。</li>
	<li>洗涤细胞三次和处理使用1毫升0.25% 胰蛋白酶在37° c 为10分钟去除部分分离的细菌。</li>
	<li>室温下20分钟, 在 PBS 中固定2.5% 的粉煤灰。</li>
	<li>用 PBS 冲洗 3x, 用流式细胞仪分析。</li>
</ol>8. 化卡介苗在巨噬细胞内的细胞内贩运
<ol><li>
荧光显微镜
	<ol>
<li>种子 3 x 105生264.7 巨噬细胞在片的 24-井板上。允许单元格在37° c 和 5% CO2的夜间粘附。</li>
		<li>在37° c 和 5% CO2的维持培养基中, 以分枝杆菌 (莫伊 10:1) 感染巨噬细胞, 4 至 24 h。</li>
		<li>清洗细胞和处理去除附着的表面, 不摄入细菌, 如步骤7.5。</li>
		<li>在 pbs 中, 在室温下20分钟修复2.5% 只粉煤灰中的感染细胞, 然后用 pbs 3 冲洗。</li>
		<li>Permeabilize 细胞在封闭/性缓冲 (0.1% 海卫 X-100, 3% BSA 在 PBS) 在室温下20分钟。<ol>
<li>使用特定的兴趣抗体 (例如, 兔抗素 Ab) 在10微克/毫升, 在性缓冲20分钟在室温下其次抗体 (如, FITC 山羊抗兔 IgG) 20 分钟。</li>
		</ol>
</li>
		<li>冲洗细胞3x 与 PBS, 一旦与水, 并在幻灯片上安装10μ l 水的安装介质, 以尽量减少荧光漂白。</li>
		<li>使用带有 63 x/1.4 计划-色差目标的荧光显微镜, 通过数字共聚焦显微镜检查幻灯片。使用与显微镜软件耦合的数码相机记录图像。</li>
	</ol>
</li>
	<li>
免疫染色与电镜
	<ol>
<li>种子 2 x 106生264.7 巨噬细胞在 6-井板中。</li>
		<li>用4% 的粉煤灰固定在室温下4小时的卡介苗感染的巨噬细胞。</li>
		<li>用 PBS 洗两次样品。</li>
		<li>在4% 低熔点琼脂糖中嵌入样品, 在70% 乙醇中脱水。</li>
		<li>将样品转移到丙烯酸树脂和聚合50° c。</li>
		<li>用切片切割出 60 nm 剖面, 并在镍网格上收集剖面。</li>
		<li>用10微克/毫升素抗体在室温或4° c 夜间在孵化溶液 (PBS 和 0.1% BSA) 中标记样品, 然后用孵化液金-共轭 F (ab)2超小山羊抗兔 IgG (1/20) 在室温下为1小时温度在孵化溶液中。</li>
		<li>用蒸馏水冲洗切片, 2% 戊二醛染色, 再冲洗, 晾干, 用电子显微镜检查。</li>
	</ol>
</li>
</ol>9. 动物免疫和器官加工
注: 所有步骤应在生物安全柜中完成。
<ol><li>通过化和蛋白质涂层细菌通过 271/2G 针10次, 使单细胞悬浮。</li>
	<li>
采取 1 x 106细菌在100μ l 无内毒素 PBS 和免疫雌性 C57BL/6 小鼠 (I-ab, H-2Kb, 5-6 星期老) 皮下的颈背。
	<ol>
<li>单独注射100μ l 的 PBS 控制小鼠。</li>
	</ol>
</li>
	<li>
安乐免疫小鼠20天后经 CO2吸入后经宫颈脱位后免疫接种。
	<ol>
<li>分离脾脏并将其转化为 RPMI 介质。</li>
	</ol>
</li>
	<li>
通过一个5毫升注射器柱塞的70µm 细胞过滤器捣碎脾脏。
	<ol>
<li>用5毫升的 RPMI 洗。离心机 (800 x g, 3 分钟) 以隔离单个单元悬浮。</li>
		<li>用小鼠生物素阳性选择试剂盒和 biotin-Ter119/Erythroid 细胞抗体消耗红细胞。</li>
		<li>离心和重新悬浮细胞在10毫升完全 RPMI (10% FCS, 1% l-谷氨酰胺, 1% 青霉素, 1% 链霉素, 和50μ m 2-我)。</li>
	</ol>
</li>
</ol>10. I-Ab聚丙烯染色, 以确定抗原特异的 CD4+ t 细胞和细胞内细胞因子染色的频率, 以确定在免疫动物中释放细胞因子的抗原特异性 t 细胞的频率
<ol><li>
聚丙烯染色
	<ol>
<li>用 PE 共轭 I-Ab-卵子323-339控制和免疫小鼠 (~ 20 x 106细胞) 脾染色体 (1/12.5 稀释) 为1小时, 在37° c 在装订缓冲器 (PBS 与 2% FCS 和0.1% 南3)。</li>
		<li>添加 AF647 CD4 Ab (1:25) 和 7-反倾销 (检测死细胞), 以脾在室温下20分钟。</li>
		<li>用流式细胞仪分析样品。</li>
		<li>在 CD4 阳性区域获得50万事件, 以确定聚丙烯阳性事件的频率。 注: 总脾是由 SSC/FSC 斑点杂交定义, 活细胞通过排除 7-反倾销阳性事件, 和 CD4 子集通过浇 AF647 阳性事件。</li>
	</ol>
</li>
	<li>
抗原特异性细胞因子释放 T 细胞的频率
	<ol>
<li>将大约 1 x 107脾转换为4毫升的完全 RPMI 介质, 在六-井板中有或没有重组卵 (10 µg/毫升) 并孵育 16 h。</li>
		<li>治疗细胞与 Brefeldin A (1:1, 000) 为另外的 5 h。</li>
		<li>用 PBS 洗涤, 并受细胞内细胞因子染色, 如下所示:<ol>
<li>在室温下为30分钟的 PE-CD8 或 PE-Cy7-CD4 Ab (1:50 在装订缓冲器中) 染色细胞样品。</li>
			<li>室温下4% 粉煤灰固定20分钟。</li>
			<li>Permeabilize 使用性解决方案, 根据制造商的指示。</li>
			<li>用 AF647-conjugated 干扰素抗体 (1:50) 在室温下20分钟清洗细胞和标签。</li>
			<li>洗涤细胞和受流式细胞仪分析如上文所述。</li>
		</ol>
</li>
	</ol>
</li>
</ol>

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<li>Bubb, M. O., Green, F., Conradie, J. D., Tchernyshev, B., Bayer, E. A., Wilchek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Natural+antibodies+to+avidin+in+human+serum%5BTitle%5D)+AND+%22ImmunolLett%22%5BJournal%5D)">Natural antibodies to avidin in human serum.</a> ImmunolLett. 35 (3), 277-280 (1993).</li>
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</ol>

作者没有什么可透露的。

我们在这项研究中报告了一种非遗传的方法快速有效地显示外源性蛋白在 BCG 表面, 以增加任何特定的抗原或特定的功能性质, 预期有效地提高细菌的免疫原性。我们证明, BCG 细胞表面可以很容易化的瞬时表面装饰与素融合蛋白。总的过程可以在2小时内完成, 而遗传转化和阳性克隆的选择需要2到3月的时间滞后。表面改性不影响细菌的生长和存活。用替代抗原卵细胞修饰的细菌能够将抗原传递到抗?...

已经提出了各种战略来取代目前的结核疫苗 bcg, 包括蛋白质佐剂系统、病毒向量技术、减毒活的m tb菌株以及基因改造的卡介苗菌株, 以引入基因过度表达 bcg 抗原, 在感染1或结核分枝杆菌特异抗原在 bcg2中没有充分表达。然而, 基因工程面临着许多障碍, 包括安全级别不确定、耗时过程和表达载体效率低下4,5。关于改进 BCG, 需要另一种方法来提高免疫原性, 而不需要不确定的基因交替。
在这项研究中, 我们介绍了一种新的策略, 显示在 BCG 细胞表面感兴趣的重组蛋白, 这是基于已知的高亲和性素与生物素的相互作用。这种方法允许快速和可重现的素融合蛋白在表面化卡介苗, 这有助于广泛的操作 bcg, 以达到最大的提高其功效, 同时保持其优良的安全性记录, 观察了几十年的使用。
素亲和性为生物素是极端高 ( Kd = 10−15 M ) 和一旦形成 , 生物素 - 素复合体是非常稳定的 , 并且在变性情况下只可能扰6。然而, 这种类型的相互作用作为一种基因转移方法的替代, 长期而可逆的显示重组蛋白是必需的。因此, 我们在这里介绍了一个低亲和力单体素 (Kd = 107 M), 这将导致从表面修饰的 BCG 在抗原呈递细胞内摄取的蛋白质的可逆释放。为了提供一个概念的证明, 我们测试了这种方法使用单体素嵌合体蛋白对应的代理抗原从蛋白 (卵子)7,8。结果表明, bcg 细胞表面可以方便、快速地用单体素融合蛋白进行修饰, 并且这种与 bcg 表面的结合是稳定和可重复的, 没有细菌生长和生存的可察觉的变化。此外, 我们发现, bcg 装饰与单体素融合与卵子 (AviOVA) 可以诱导免疫反应类似的 bcg 诱导的基因表达相同的抗原都在体外和在体内。这一技术的可逆显示蛋白质的细菌表面是一个有效的替代传统的基因转移方法, 可以提供一个平台的广泛操作卡介苗和进一步应用在疫苗发展.

<table><tbody><tr><td>Endotoxin-free RPMI 1640</td><td>StemCell Technologies&amp;nbsp;</td><td>36750</td><td></td></tr><tr><td>Sulfo-NHS SS biotin</td><td>Thermo Fisher&amp;nbsp;</td><td>21328</td><td></td></tr><tr><td>FITC-conjugated streptavidin</td><td>Sigma-Aldrich</td><td>S3762</td><td></td></tr><tr><td>Phycoerythrin (PE)-conjugated I-A&lt;sup&gt;b&lt;/sup&gt;-OVA&lt;sub&gt;323-339&lt;/sub&gt; tetramer&amp;nbsp;</td><td>MBL International&amp;nbsp;</td><td>TS-M710-1</td><td></td></tr><tr><td>7-AAD</td><td>BD Pharmingen</td><td>559925</td><td></td></tr><tr><td>TALON polyhistidine-Tag purification resin&amp;nbsp;</td><td>Clontech</td><td>635501</td><td></td></tr><tr><td>Alexa Fluor (AF) 647 conjugated rat anti-mouse CD4</td><td>BD Bioscience&amp;nbsp;</td><td>557681</td><td></td></tr><tr><td>AF647 rat anti-mouse IFN-g</td><td>BD Bioscience&amp;nbsp;</td><td>557735</td><td></td></tr><tr><td>AF647 rat anti-mouse I-A/I-E</td><td>BD Bioscience&amp;nbsp;</td><td>562367</td><td></td></tr><tr><td>PeCy7 rat anti-mouse CD4</td><td>BD Bioscience&amp;nbsp;</td><td>552775</td><td></td></tr><tr><td>PE rat anti-mouse CD8 Ab</td><td>BD Bioscience&amp;nbsp;</td><td>561095</td><td></td></tr><tr><td>AF 647 rat anti-mouse H-2k&lt;sup&gt;b&lt;/sup&gt;</td><td>BD Bioscience</td><td>562832</td><td></td></tr><tr><td>FITC-conjugated goat anti rabbit antibody</td><td>Thermo Fisher</td><td>31635</td><td></td></tr><tr><td>AF 647 rat anti-mouse CD4</td><td>BD Bioscience&amp;nbsp;</td><td>557681</td><td></td></tr><tr><td>Ultra-small gold-conjugated goat anti-rabbit IgG</td><td>Electron Microscopy Sciences</td><td>25100</td><td></td></tr><tr><td>Middlebrook 7H9 broth</td><td>BD Diagnostic Systems</td><td>271310</td><td></td></tr><tr><td>OADC</td><td>BD Diagnostic Systems</td><td>B11886</td><td></td></tr><tr><td>Tween 80</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>RAW 264.7 murine macrophage cell lines</td><td>American Type Culture Collection</td><td></td><td></td></tr><tr><td>pDEST17 plasmid&amp;nbsp;</td><td>Invitrogen</td><td>11803012</td><td></td></tr><tr><td>pUC57-OVA plasmid&amp;nbsp;</td><td>GenScript</td><td>SD1176</td><td></td></tr><tr><td>BP clonase&amp;nbsp;</td><td>Invitrogen</td><td>11789020</td><td></td></tr><tr><td>LR clonase&amp;nbsp;</td><td>Invitrogen</td><td>11791043</td><td></td></tr><tr><td>pDONR221 plasmid</td><td>Invitrogen</td><td>12536017</td><td></td></tr><tr><td>Ni-NTA columns&amp;nbsp;</td><td>Qiagen</td><td>31014</td><td></td></tr><tr><td>Pierce protein concentrators&amp;nbsp;</td><td>Thermo Fisher&amp;nbsp;</td><td>88527</td><td></td></tr><tr><td>Flurosave</td><td>Calbiochem-Novabiochem</td><td>345789</td><td></td></tr><tr><td>Axioplan II epifluorescence microscope</td><td>Carl Zeiss Inc</td><td></td><td></td></tr><tr><td>CCD digital camera&amp;nbsp;</td><td>Retiga EX, QImaging</td><td></td><td></td></tr><tr><td>Tecnai G2 200kV electron microscope&amp;nbsp;</td><td>FEI Company</td><td>G2 200Kv&amp;nbsp;</td><td></td></tr><tr><td>70&amp;mu;m Falcon cell strainer&amp;nbsp;</td><td>Thermo Fisher&amp;nbsp;</td><td>87712</td><td></td></tr><tr><td>EasyStep mouse biotin positive selection kit&amp;nbsp;</td><td>StemCell</td><td>18556</td><td></td></tr><tr><td>biotin-Ter119/Ertyroid cells antibody</td><td>BioLegend</td><td>116203</td><td></td></tr><tr><td>Brefeldin A</td><td>BD Pharmingen</td><td>555029</td><td></td></tr><tr><td>Cytofix/Cytoperm kit&amp;nbsp;</td><td>BD Pharmingen</td><td>554714</td><td></td></tr><tr><td>Bright-Glo Luciferase assay system</td><td>Promega</td><td>e2620</td><td></td></tr><tr><td>Turner Biosystem luminometer&amp;nbsp;</td><td>Promega</td><td>TD-20/20</td><td></td></tr><tr><td>Leica EM UC6 microtome</td><td>&amp;nbsp;Leica Microsystems</td><td>UC6</td><td></td></tr><tr><td>Novalyphe NL 500 freeze dryer</td><td>Savant Instruments</td><td>&amp;nbsp;NL 50</td><td></td></tr><tr><td>Wheaton boroscilicate glass vials</td><td>Wheaton</td><td>&amp;nbsp;VWR 66011-675</td><td></td></tr></tbody></table>

expression of exogenous antigens in the mycobacterium bovis bcg vaccine via non-genetic surface decoration with the avidin-biotin system

肺结核 (tb) 是一种严重的传染性疾病, 唯一可用的疫苗是牛牛卡介苗 Guérin (BCG) 是安全和有效的保护儿童的严重结核脑膜炎和某些形式的传播结核, 但未能保护肺结核, 这是最普遍的疾病形式。有前途的战略, 以改善 BCG 目前依赖于其转化的基因编码免疫m 结核)特异抗原和/或互补基因编码共同因素, 将刺激抗原呈现单元格。这些方法的主要局限性包括效率低、稳定性低、表达载体安全程度不确定等。在这项研究中, 我们提出了一种替代方法的疫苗改进, 其中包括卡介苗与外源蛋白的利益的表面上的细菌, 而不是转化与质粒编码相应的基因。首先, 在标准的大肠杆菌表达系统中, 有兴趣的蛋白质与单体素融合, 然后用来装饰化 BCG 的表面。用蛋白抗原修饰的 BCG 表面进行动物实验, 表明该修饰菌完全免疫, 能够诱导特定的 T 细胞反应。总而言之, 这里所提供的数据强烈支持一种新的、有效的方法来重塑目前的 BCG 疫苗, 取代了传统的与外源核酸互补的方法。

采用上述一般程序, 研究了 BCG 表面法和替代抗原卵修饰的可行性。经改良的卡介苗的免疫原性被测试在体内。细菌表面易于标记的生物素快速显示素嵌合抗原没有任何可察觉的变化的细菌表型。通过抗原表达细胞有效地摄取了经过改良的卡介苗, 并能诱导小鼠的特定卵子免疫应答。
单体素融合质粒和蛋白质的生成...

Watch this Scientific Journal Video about Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System at JoVE.com

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

提出了一种新的细菌表面快速抗原显示技术, 它涉及表面法, 其次是暴露于与单体素融合的感兴趣的蛋白质。用选定抗原载入卡介苗, 成功地提高了其免疫原性, 表明表面修饰可以取代传统的遗传方法。

非遗传表面修饰的素-生物素系统在牛分枝杆菌卡介苗中的外源抗原表达

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

expression-exogenous-antigens-mycobacterium-bovis-bcg-vaccine-via-non

Research

JoVE Journal

Immunology and Infection

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

9.4K 次观看.  University of British Columbia. 提出了一种新的细菌表面快速抗原显示技术, 它涉及表面法, 其次是暴露于与单体素融合的感兴趣的蛋白质。用选定抗原载入卡介苗, 成功地提高了其免疫原性, 表明表面修饰可以取代传统的遗传方法。

视频: Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

科研

免疫与感染

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

副结核分枝杆菌在自身免疫性脑脊髓炎中的免疫原性增强

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in complete Freund&#39;s adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund&#39;s adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10.
M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne&#39;s disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP)&#160;strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.

作者聚焦: 副结核分枝杆菌在增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原性中的辅助活性  

Here, we present an alternative protocol to actively induce experimental autoimmune encephalomyelitis in C57BL/6 mice, using the immunogenic epitope myelin oligodendrocyte glycoprotein (MOG)35-55 suspended in incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis.

副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in ...

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites, lipids, proteins, and nucleic acids, several groups have evaluated either the native or recombinant versions of bEVs for their protective potency as subunit vaccine candidates. Unlike native EVs, recombinant EVs are molecularly engineered to contain one or more immunogens of interest. Over the last decade, different groups have explored diverse approaches for generating recombinant bEVs. However, here, we report the design, construction, and enrichment of recombinant mycobacterial EVs (mEVs) in mycobacteria. Towards that, we use Mycobacterium smegmatis (Msm), an avirulent soil mycobacterium as the model system. We first describe the generation and enrichment of native EVs of Msm. Then, we describe the design and construction of recombinant mEVs that contain either mCherry, a red fluorescent reporter protein, or EsxA (Esat-6), a prominent immunogen of Mycobacterium tuberculosis. We achieve this by separately fusing mCherry and EsxA N-termini with the C-terminus of a small Msm protein Cfp-29. Cfp-29 is one of the few abundantly present proteins of MsmEVs. The protocol to generate and enrich recombinant mEVs from Msm remains identical to the generation and enrichment of native EVs of Msm.

This protocol details the enrichment of native mycobacterial extracellular vesicles (mEVs) from axenic cultures of Mycobacterium smegmatis (Msm) and how mCherry (a red fluorescent reporter)-containing recombinant MsmEVs can be designed and enriched. Lastly, it verifies the novel approach with the enrichment of MsmEVs containing the EsxA protein of Mycobacterium tuberculosis.

富集分枝杆菌的天然和重组细胞外囊泡

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, ...

使用 Micro-CFU 方法加速结核病测定

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during the 2019-2021 pandemic. The disease is caused by the bacterium Mycobacterium tuberculosis (M.tb). The Mycobacterium bovis strain Bacillus Calmette-Gu&#233;rin (BCG), the only TB vaccine, is the oldest licensed vaccine in the world, still in use. Currently, there are 12 vaccines in clinical trials and dozens of vaccines under pre-clinical development. The method of choice used to assess the efficacy of TB vaccines in pre-clinical studies is the enumeration of bacterial colonies by the colony-forming units (CFU) assay. This time-consuming assay takes 4 to 6 weeks to conclude, requires substantial laboratory and incubator space, has high reagent costs, and is prone to contamination. Here we describe an optimized method for colony enumeration, the micro-CFU (mCFU), that offers a simple and rapid solution to analyze M.tb vaccine efficacy results. The mCFU assay requires tenfold fewer reagents, reduces the incubation period threefold, taking 1 to 2 weeks to conclude, reduces lab space and reagent cost, and minimizes the health and safety risks associated with working with large numbers of M.tb. Moreover, to evaluate the efficacy of a TB vaccine, samples may be obtained from a variety of sources, including tissues from vaccinated animals infected with Mycobacteria. We also describe an optimized method to produce a unicellular, uniform, and high-quality mycobacterial culture for infection studies. Finally, we propose that these methods should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB.

作者聚焦:优化 CFU 测定以有效评估结核病疫苗功效和抗原呈报分析 

The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

用于结核病疫苗功效评估的微集落形成单位测定

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...

非遗传表面修饰的素-生物素系统在牛分枝杆菌卡介苗中的外源抗原表达

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一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

富集分枝杆菌的天然和重组细胞外囊泡

用于结核病疫苗功效评估的微集落形成单位测定

用于结核病疫苗功效评估的微集落形成单位测定

用于结核病疫苗功效评估的微集落形成单位测定