Name: expression of exogenous antigens in the mycobacterium bovis bcg vaccine via non-genetic surface decoration with the avidin-biotin system
Uploaded: 2018-01-31T13:00:00
Description: 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

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...

<ol>
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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.

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</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 ...

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

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

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

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

Culturing Microglia from the Neonatal and Adult Central Nervous System

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 ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

在FDA批准的Vero细胞疫苗开发产生重组沙粒

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

从新型H7N9流感病毒利用杆状病毒表达系统的功能重组血凝素和神经氨酸酶蛋白的表达

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcÃƒÅ½Ã‚ÂµRI

的发展<em&gt;在体外</em&gt;模型研究的交互系统<em&gt;雅科仕</em&gt;<em&gt; caballus</em&gt;以其高亲和力受体了FcεRI的IgE

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-GuÃƒÆ’Ã†'Ãƒâ€šÃ‚Â©rin

基于CFSE-试验鼠皮肤树突状细胞的迁移习进引流淋巴结在与感染<em&gt;牛分枝杆菌</em&gt;卡介苗

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

纯化膜隔间跨演示文稿中的外源抗原内质网关联降解

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...