抗人纤维蛋白单链抗体-链亲和素融合蛋白的稳定性及对动物纤维蛋白的识别

Stability of anti human fibrin single chain antibody: core streptavidin fusion protein and interaction with other animal fibrin

目的：探索抗人纤维蛋白单链抗体链亲合素融合蛋白的应用前景。方法：构建抗人纤维蛋白单链抗体ｓｃＦｖ８Ｅ５基因与链亲和素基因的重组表达载体ｐＳＴＥ２８Ｅ５ ，表达的融合蛋白可特异性识别人纤维蛋白，同时有生物素的结合位点。转化ＪＭ１０９ 后用ＩＰＴＧ 诱导表达，ＥＬＩＳＡ 观察融合蛋白的稳定性。结果：抗人纤维蛋白单链抗体链亲合素融合蛋白稳定性好，可耐受至少２ ｗ ，４ ℃保存、数次反复冻融或一定时间的３７ ℃孵育。它能与大鼠和豚鼠的纤维蛋白结合，而与猪纤维蛋白反应不明显。结论：抗人纤维蛋白单链抗体链亲和素融合蛋白适用于大鼠或豚鼠血栓病模型，是临床前研究的有用材料。

Objective: To explore the clinical use of anti human fibrin single chain antibody: core streptavidin fusion protein in the future Methods:The author have constructed an anti human fibrin single chain antibody: core streptavidin fusion protein by combining the core streptavidin sequence with the anti human fibrin scFv 8E5 gene The fusion protein expressed by the vector pSTE2 8E5 contained the biotin binding site which could be used for direct detection of fibrin in ELISA with biotinylated horseradish peroxidase The transformed E coli JM109 cells were propagated and induced by IPTG Results:This fusion protein was rather stable; it could tolerate 4℃ storage for at least two weeks, freezing/thawing several times or incubation at 37℃ for a certain time This fusion protein was also able to bind to rat or guinea pig fibrin, but not to pig fibrin Conclusion:Anti human fibrin single chain antibody: core streptavidin fusion protein could be taken for developing reliable experimental animal models for the study of thrombus imaging and the targeting of thrombolytic agents