摘要
目的优化表达条件,获取有生物活性的特异性抗肝癌单链抗体。方法以ITPG诱导大肠杆菌HB2151表达可溶性抗肝癌单链抗体;利用HiTrap Anti-ETag亲和层析柱对阳性克隆表达的单链抗体进行纯化;纯化后的单链抗体进行SDS-聚丙烯酰胺凝胶电泳,以Bradford检测法测定其浓度,ELISA法及免疫组化法鉴定其生物学活性;结果在培养基中加入0.4M蔗糖,以IPTG 1mmol/L于30℃诱导12h,抗肝癌scFv可溶性表达量较多,纯化后可溶性抗肝癌scFv含量为325μg/L。纯化的抗肝癌scFv与肝癌组织、肝癌细胞抗原特异性结合,与肝硬化、正常肝组织无阳性结合。结论成功获得具有生物活性的特异性抗肝癌单链抗体,为肝癌靶向诊断及治疗的开发与应用奠定了基础。
[Objective] To obtain the functional single-chain Fv antibody against hepatocellular carcinoma. [Methods] After induced by IPTG, the soluble scFvs against HCC were expressed in the bacteria periplasm of E. coli HB2151. Using HiTrap Anti-E Tag column, the scFvs were purified by affinity chromatography. SDS-polyacrylamlde gel electrophoresis of purified scFvs was analyzed. The consistencies of purified scFvs were measured by Bradford method. The bioactivities of purified scFv were identified by ELISA and immunohistochemistry. [Result] After 12 h induced by 1 mmol IPTG at 30℃ with 0.4 M sucrose, the amount of expressed sc＇Fv increased greatly. The yield of purified scFv was 325 μg/L. The purified scFv was specially binding to tumor antigen of HCC. [ Conclusions] The functional single-chain Fv antibody against hepatocellular carcinoma was achieved, which is essential of using this single-chain Fv antibody in hepatocellular carcinoma targeting diagosis and therapy.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2008年第11期1525-1528,共4页
China Journal of Modern Medicine
基金
广东省科技攻关项目(No:2006B19901014)
广东省名医工程研究项目(粤卫(2004)199号)
关键词
肝细胞癌
单链抗体
优化表达
纯化
鉴定
hepatocellular carcinoma
single-chain Fv antibody
optimized expression
purity
identification
