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首页> 外文期刊>Cellular immunology >Expression and characterization of recombinant soluble porcine CD3 ectodomain molecules: Mapping the epitope of an anti-porcine CD3 monoclonal antibody 898H2-6-15
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Expression and characterization of recombinant soluble porcine CD3 ectodomain molecules: Mapping the epitope of an anti-porcine CD3 monoclonal antibody 898H2-6-15

机译：重组可溶性猪CD3胞外域分子的表达和表征：定位抗猪CD3单克隆抗体898H2-6-15的表位

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摘要

The porcine CD3 specific monoclonal antibody 898H2-6-15 has been used in allo- and xeno-transplantation studies as a porcine CD3 marker and as an effective T cell depletion reagent when conjugated to the diphtheria toxin mutant, CRM9. A recombinant anti-porcine CD3 immuntoxin was recently developed using single-chain variable fragments (scFv) derived from 898H2-6-15. In this study, using published sequence data, we have expressed the porcine CD3 ectodomain molecules in E. coli through inclusion body isolation and in vitro refolding approach. The expressed and refolded porcine CD3 ectodomain molecules include CD3ε, CD3γ, CD3δ, CD3εγ heterodimer, CD3εδ heterodimer, CD3εγ single-chain fusion protein and CD3εδ single-chain fusion protein. These refolded porcine CD3 ectodomain molecules were purified with a strong anion exchange resin Poros 50HQ. ELISA analysis demonstrated that only the porcine CD3εγ ectodomain single-chain fusion protein can bind to the porcine CD3 specific monoclonal antibody 898H2-6-15. The availability of this porcine CD3εγ ectodomain single-chain fusion protein will allow screening for affinity matured variants of scFv derived from 898H2-6-15 to improve the recombinant anti-porcine CD3 immunotoxin. Porcine CD3εγ ectodomain single-chain fusion protein will also be a very useful reagent to study the soluble phase interaction between porcine CD3εγ and porcine CD3 antibodies such as 898H2-6-15.
机译：猪CD3特异性单克隆抗体898H2-6-15已在同种异体和异种移植研究中用作猪CD3标记物，并在与白喉毒素突变体CRM9偶联后用作有效的T细胞清除剂。最近使用衍生自898H2-6-15的单链可变片段（scFv）开发了重组抗猪CD3免疫毒素。在这项研究中，我们使用公开的序列数据，通过包涵体分离和体外重折叠方法在大肠杆菌中表达了猪CD3胞外域分子。表达和重折叠的猪CD3胞外域分子包括CD3ε，CD3γ，CD3δ，CD3εγ异二聚体，CD3εδ异二聚体，CD3εγ单链融合蛋白和CD3εδ单链融合蛋白。这些重折叠的猪CD3胞外域分子用强阴离子交换树脂Poros 50HQ纯化。 ELISA分析表明，只有猪CD3εγ胞外域单链融合蛋白可以与猪CD3特异性单克隆抗体898H2-6-15结合。此猪CD3εγ胞外域单链融合蛋白的可用性将允许筛选源自898H2-6-15的scFv的亲和力成熟变体，以改善重组抗猪CD3免疫毒素。猪CD3εγ胞外域单链融合蛋白也将是研究猪CD3εγ与猪CD3抗体（例如898H2-6-15）之间的可溶性相相互作用的非常有用的试剂。

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