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目的人源化抗人纤维蛋白单链抗体的重链，降低人抗鼠抗体反应。方法从基因文库中挑选到与鼠源抗人纤维蛋白单链抗体重链（ＶＨ－８Ｅ５）同源性７１．８％的人源免疫球蛋白ＨＵＭＨＣＨ１０９重链（ＶＨ）序列，作为人源化改造ＶＨ－８Ｅ５的框架。人工合成人源化ＶＨ－８Ｅ５片段，用连接酶连接成完整的人源化ＶＨ－８Ｅ５，构建表达载体ｐＯＰＥ１０１－ＶＨ－８Ｅ５。转化ＪＭ１０９后用异丙基－β－Ｄ－硫代半乳糖苷诱导表达。结果经聚丙烯酰胺电泳和ＥＬＩＳＡ证明表达产物相对分子质量３０×１０３左右，有特异性识别和结合人纤维蛋白能力，是亲本抗体ＳｃＦｖ－８Ｅ５活性的４／５左右。人源化ＳｃＦｖ－８Ｅ５表达产量为转化菌总蛋白的１１．２％。结论人源化ＶＨ－８Ｅ５仍具有特异性识别人纤维蛋白的活性，ＳｃＦｖ－８Ｅ５重链的人源化基本获得成功
Objective Humanized anti-human fibrin scFv heavy chain, reducing human anti-mouse antibody response. Methods Human immunoglobulin HUMHCH109 heavy chain (VH) sequence with a homology of 71.8% with the murine anti-human fibrin single chain antibody heavy chain (VH-8E5) was selected from the gene library as a humanized VH-8E5 framework. Humanized VH-8E5 fragment was synthesized and ligated into intact humanized VH-8E5 to construct expression vector pOPE101-VH-8E5. JM109 transformed with isopropyl-β-D-thiogalactoside induced expression. Results The polyacrylamide gel electrophoresis and ELISA showed that the relative molecular mass of the expressed product was about 30 × 103. The ability to specifically recognize and bind human fibrin was about 4/5 of the activity of the parental antibody, ScFv-8E5. The expression yield of humanized ScFv-8E5 was 11.2% of the total transformed cells. Conclusions Humanized VH-8E5 still has the activity of specifically recognizing human fibrin, and the humanization of ScFv-8E5 heavy chain has basically succeeded