【摘要】 目的:表达及纯化抗去唾液酸糖蛋白受体(ASG-PR)的单链抗体的可溶性,并测定其亲和常数。方法:用噬菌体C1克隆感染E.coliHB2151,挑取单个菌落接种于2×TY培养基中,于37℃震荡培养过夜。将培养物作1∶100稀释并转种后,用终浓度为0.25、0.5、1.0mmol/L的IPTG,分别在37℃、25℃和20℃下诱导表达过夜。取其培养上清,用饱和硫酸铵沉淀后,以120g/LSDS-PAGE分析。另外,将饱和硫酸铵沉淀物用30mLPBS重新溶解、透析除盐后,用Ni2+螯合柱进行纯化,再以120g/LSDS-PAGE鉴定纯化scFvC1的纯度。用非竞争酶免疫法测定scFv的亲和常数。结果:用0.5mmol/LIPTG在25℃诱导过夜,表达的scFvC1的量较多,其相对分子质量(Mr)约为28000,以可溶性的形式存在于培养基中。通过Ni2+亲和柱纯化后scFvC1的纯度在95%以上,产量约为0.8mg/L。scFv的亲和常数为(2.31±0.36)×10-7mol/L。结论:以筛选的C1噬菌体感染E.coliHB2151后可表达低亲和力的可溶性scFv,对肝癌的基因治疗具有潜在的应用价值。更多还原

【Abstract】 AIM: To express and purify a single chain Fv antibody (scFv) C1 against human hepatic asialoglycoprotein receptor (ASGPR) and to determine affinity constant of the purified scFv C1. METHODS: The specific anti-ASGPR phage clone C1 was transfected into E.coli HB2151. The single colony was chosen to be inoculated into 2×TY medium and shaken (250 r/min) overnight at 37℃. After 1 in 100 dilution in 2×TY medium and induced for secreted expression, scFv C1 was induced at different concentrations (0.25, 0.5 and 1.0 mmol/L IPTG) overnight at 37℃, 25℃ and 20℃, respectively. The supernatant was precipitated with saturated ammonium sulfate and its sediment was analyzed by SDS-PAGE. In addition, the sediment was resuspended in 30 mL PBS and dialyzed against PBS overnight at 4℃. The expressed scFv C1 was purified by Ni~ 2+ chelating HiTrap HP column and the purity of the purified scFv C1 was identified by SDS-PAGE. Then affinity constant of scFv C1 was determined by noncompetitive enzyme immunoassay. RESULTS: After induced with 0.5 mmol/L IPTG overnight at 25℃, the amount of expressed scFv C1 increased greatly and its M_r was about 28 000, and it existed in culture supernant in soluble form. The purity of scFv C1 by nickel-agarose column was above 95% and its yield was about 0.8 mg/L. The affinity constant of the purified scFv C1 was confirmed to be (2.31±0.36)×10~ -7 mol/L. CONCLUSION: The E.coli HB2151 infected with phage C1 clones may express soluble scFv C1 with low affinity, which has potential applications to gene therapy of hepatocellular carcinoma.更多还原