【摘要】 从SARS免疫抗体库获得的一株抗SARSCoV人源单链抗体H12,亟待鉴定。为了快速制备大量具有生物活性的单链抗体H12,构建了pET28aH12原核高表达载体,表达量占菌体总蛋白质30%以上。采用稀释复性和分子筛柱复性两种方法对包涵体蛋白进行复性与纯化,结果显示两种方法都能使得单链抗体复性。与稀释复性法相比,柱复性效果更好,其抗原结合活性是稀释复性法的1.51倍。柱复性后的单链抗体亲和力测定的解离常数Kd为73.5nmolmL。为进一步研究单链抗体H12的功能奠定了基础。更多还原

【Abstract】 A novel human ScFv H12 against SARS-CoV has been selected from a SARS immune library. In order to produce a large amount of ScFv H12, pET28a-H12 expression vector was constructed and ScFv H12 was expressed at yield about 30% of total proteins in E.coli. Here two different refolding procedures were used to refold ScFv H12 from inclusion body: gel filtration chromatography and dilution. The results showed that ScFv H12 could be efficiently refolded by both procedures. However, the refolding via gel filtration was 1.5 time more effective than that of dilution. The affinity of ScFv H12 to SARS-CoV virion was detected as Kd=73.5nmol/mL.更多还原