Pathogenesis of Shigella diarrhea. IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies
Pathogenesis of Shigella diarrhea. IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies
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志贺氏菌腹泻的发病机制。
DOI：
--
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发表时间：
1984
影响因子：
15.3
通讯作者：
M. Jacewicz
中科院分区：
文献类型：
--
作者：
A. Donohue‐Rolfe;G. T. Keusch;Clark Edson;David THORLEY;M. Jacewicz
设计了一个简单的纯化方案，志贺菌细胞毒素，导致高产率（约50%）和比活性增加1,300倍，与初始的粗细菌细胞裂解物相比。纯化的毒素在结扎的兔回肠袢中具有肠毒性，当注入小鼠腹腔时具有神经毒性。细胞毒素和肠毒素的比活性测定表明，这两种毒性在分级过程中共纯化。在十二烷基硫酸钠凝胶电泳上，毒素以两个多肽亚基迁移，一个为32，000 mol wt的A亚基和一个为6，500 mol wt的B亚基。化学交联实验表明，该毒素是由一个A和五个B亚基组成的复合物，分子量为64，000。制备多克隆兔抗毒素和抗亚单位B抗血清以及亚单位特异性小鼠单克隆抗体。与毒素一起预孵育的所有抗体中和HeLa细胞单层中的细胞毒性作用。相反，只有A亚基特异性抗体能够中和预先结合到HeLa细胞表面的毒素。B亚单位的抗体也抑制了94%或更多的125 I标记的毒素与这些细胞的结合。这些数据表明，B亚基参与志贺氏菌毒素与细胞表面的结合。
A simple purification scheme for shigella cytotoxin was devised, resulting in high yields (approximately 50%) and a 1,300-fold increase in specific activity compared with the initial crude bacterial cell lysate. The purified toxin was enterotoxic in ligated rabbit ileal loops and neurotoxic when injected into the peritoneal cavity of mice. Measurement of specific activity of cytotoxin and enterotoxin demonstrated that these two toxicities copurify during the fractionation procedure. On sodium dodecyl sulfate gel electrophoresis, the toxin migrated as two polypeptide subunits, an A subunit of 32,000 mol wt and a B subunit of 6,500 mol wt. Chemical cross-linking experiments demonstrate that the toxin is a complex consisting of one A and five B subunits with a molecular weight of 64,000. Polyclonal rabbit anti-toxin and anti-subunit B antisera were produced as well as subunit-specific mouse monoclonal antibodies. All antibodies preincubated with toxin neutralized cytotoxic effects in HeLa cell monolayers. In contrast, only A subunit-specific antibodies were able to neutralize toxin prebound to the HeLa cell surface. Antibody to the B subunit also inhibited binding of 125I-labeled toxin to these cells by 94% or more. These data demonstrate that the B subunit is involved in shigella toxin binding to the cell surface.
志贺氏菌腹泻的发病机制。
DOI：
10.1093/infdis/148.5.844
发表时间：
1983
期刊：
The Journal of infectious diseases
影响因子：
--
作者：
Jacewicz,M;Keusch,GT
通讯作者：
Keusch,GT
伯胺和氯喹抑制志贺氏菌毒素的细胞毒性反应，并允许后期抗体拯救毒素处理的细胞。
DOI：
10.1016/0006-291x(84)90689-2
发表时间：
1984
影响因子：
3.1
作者：
Keusch,GT;Jacewicz,M
通讯作者：
Jacewicz,M