【摘要】 目的：人源化抗人纤维蛋白单链抗体，降低人抗鼠抗体反应。方法：从人免疫球蛋白基因数据库中挑选到与鼠源抗人纤维蛋白单链抗体ｓｃＦｖ－８Ｅ５重链（ＶＨ－８Ｅ５）和轻链（ＶＬＳＭ）同源性最高的序列，作为人源化改造的框架，其中植入ｓｃＦｖ－８Ｅ５的互补决定区。人工合成其ＤＮＡ片段，用ＤＮＡ连接酶连接成完整的人源化ＶＨ－８Ｅ５和ＶＬ－８Ｅ５，构建表达载体，转化ＪＭ１０９后用ＩＰＴＧ诱导表达，ＥＬＩＳＡ测定其抗原结合活性。结果：表达产物分子量３０ｋＤ左右，人源化ｓｃＦｖ－８Ｅ５与亲本抗体ｓｃＦｖ－８Ｅ５抗原结合活性无明显差别，可耐受至少２ｗ，４℃保存，数次反复冰融或一定时间的３７℃孵育。结论：人源化ｓｃＦｖ－８Ｅ５具有特异性识别人纤维蛋白的活性，鼠源ｓｃＦｖ－８Ｅ５的人源化基本获得成功。更多还原

【Abstract】 Objective: In order to reduce the human anti-mouse antibody response, anti-human fibrin scFv-8E5 has been humanized.Methods:The authors constructed a "humanized" antibody fragments by grafting the complementarity-determining regions (CDRs) of themurine anti-human fibrin scFv-8E5 to human framework regions which showed maximal homology to the scFv-8E5 sequence. The humanizedVH-8E5 and VL-8E5 DNA fragments were then synthesized and ligated into the intact heavy and light voriable variable regions respectively.The expression vectors were constructed by substituting the variable regions of scFv-8E5 with the humanized variable regions. The transformed E. coliJM109 cells were propagated and induced by IPTG. Results: Expression Products were found in the total cell extracts and periplasmic space bySDS-PAGE and ELISA. It was a 30 kD single-chain fragment with human fibrin-binding specificity. The humanized scFv-8E5 showed the samefibrin-binding activity as that of the murine scFv-8E5. It could tolerate 4c storage for at least two wall, freezing/thawing several times or incubation at 37C for a certain the. Conclusions:The humanized SCFv-8E5 showed antigen-binding capacity of the parental scFv. It is usefulrecagents for the research of clinical use.更多还原