摘要
目的制备抗人凝血因子Ⅷ(FⅧ)C1C2区单克隆抗体(mAb),并对其进行生化和生物学鉴定。方法采用基因重组技术表达FⅧ-C1C2区蛋白(rFⅧ-C1C2),以rFⅧ-C1C2免疫8周龄Balb/C小鼠,小鼠脾脏细胞与小鼠骨髓瘤细胞融合后,ELISA法筛选阳性克隆,制备稳定分泌抗rFⅧ-C1C2mAb的细胞株,并对mAb进行生化和免疫学鉴定。结果获得一株抗rFⅧ-C1C2区单克隆抗体阳性克隆2B11,命名为SZ-133。琼脂糖双扩散鉴定为IgG1类,Westernblot法证实SZ-133可与rFⅧ-C1C2区蛋白及重组的全长FⅧ特异性结合,但不影响FⅧ的凝血活性。结论表达了rFⅧ-C1C2,并制备出了抗人凝血因子Ⅷ的单克隆抗体SZ-133,它能特异识别血浆及重组的FⅧ,有可能用于基础和临床应用研究。
Objective To develop monoclonal antibody(mAb)anti-FⅧ C1C2 domain of human and characterize its biochemical and biological functions.Methods FactorⅧ-C1C2 protein was expressed by prokaryotic expression system.Balb/c mice of eight-week ages were immunized by intraperitoneal and subcutaneous injection of rFⅧ-C1C2 protein.The positive clone was screened by ELISA and identified using western-blot.Results A positive clone 2B11 designated as SZ-133 was produced,belonging to IgG1 subclass.Western blot demonstrated that SZ-133 could specifically recognized rFⅧ-C1C2 and fⅧ in human plasma.Conclusion rFⅧ-C1C2 is expressed in Ecoli.The successfully developed mAb against human FⅧ-C1C2 domain mAb can be useful for basic and clinical application.
出处
《苏州大学学报(医学版)》
CAS
北大核心
2010年第1期106-109,共4页
Suzhou University Journal of Medical Science
基金
江苏省高校自然科学基金资助项目(08KJD310008)
江苏省卫生厅"科教兴卫工程"临床医学中心血液病开放课题基金资助项目(WKF07009)
关键词
人凝血因子Ⅷ(FⅧ)C1C2区
原核表达
单克隆抗体
鉴定
human coagulation factor Ⅷ(FⅧ)C1C2 domain
prokaryotic expression
monoclonal antibody
identification