BACTERIAL EXPRESSION OF A SINGLE-CHAIN FV FRAGMENT WHICH EFFICIENTLY PROTECTS THE ACETYLCHOLINE-RECEPTOR AGAINST ANTIGENIC MODULATION CAUSED BY MYASTHENIC ANTIBODIES
BACTERIAL EXPRESSION OF A SINGLE-CHAIN FV FRAGMENT WHICH EFFICIENTLY PROTECTS THE ACETYLCHOLINE-RECEPTOR AGAINST ANTIGENIC MODULATION CAUSED BY MYASTHENIC ANTIBODIES
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DOI：
10.1002/eji.1830230816
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发表时间：
1993-08-01
影响因子：
5.4
通讯作者：
TZARTOS, SJ
中科院分区：
文献类型：
--
作者：
MAMALAKI, A;TRAKAS, N;TZARTOS, SJ
针对乙酰胆碱受体 (AChR) 主要免疫原性区域 (MIR) 的单克隆抗体 (mAb) 在诱导动物和肌肉细胞培养物中 AChR 的抗原调节方面非常有效。通过聚合酶链式反应克隆大鼠抗MIR mAb198的重组抗体片段，并在大肠杆菌中表达为可溶性单链Fv片段（scFv198）并进行亲和纯化。 DNA 测序用于定义 VH (IB) 和 VL (K2) 链基因的使用。 scFv198 被发现具有免疫学和生物活性。它对 Torpedo AChR (K(D) = 2 +/- 0.6 nm) 的结合亲和力与完整 mAb198 (K(D) = 1.8 +/- 0.6 nm) 非常相似，而对人 AChR (K(D) = 80.7 +/- 16.6 nm) 的结合亲和力比完整 mAb198 (K(D) = 21.6 +/- 16.6 nm) 低约四倍6.6 纳米）。该片段能够有效保护人类细胞培养物中的 AChR，免受完整 mAb198 或肌无力患者抗体引起的抗原调节。因此，经过适当的基因操作后，产生的 scFv198 片段可能可用于重症肌无力的治疗应用。
Monoclonal antibodies (mAb) against the main immunogenic region (MIR) of the acetylcholine receptor (AChR) are very potent in inducing antigenic modulation of the AChR in animals and in muscle cell cultures. A recombinant antibody fragment of the rat anti-MIR mAb198 was cloned by polymerase chain reaction and expressed as soluble single-chain Fv fragment (scFv198) in E. coli and affinity purified. DNA sequencing was used to define the VH (IB) and VL (K2) chain gene usage. scFv198 was found immunologically and biologically active. Its binding affinity for the Torpedo AChR (K(D) = 2 +/- 0.6 nm) was very similar with that of the intact mAb198 (K(D) = 1.8 +/- 0.6 nm) while for the human AChR (K(D) = 80.7 +/- 16.6 nm) it was about four times lower than that of the intact mAb198 (K(D) = 21.6 +/- 6.6 nm). This fragment was capable of efficiently protecting the AChR in human cell cultures, against antigenic modulation caused by the intact mAb198 or by the antibodies from a myasthenic patient. The produced scFv198 fragment is, therefore, potentially useful in therapeutic applications for myasthenia gravis after appropriate genetic manipulations.