抗肝癌单链抗体与PE38融合蛋白在大肠杆菌中的可溶性表达

Soluble expression of anti-hepatoma dsFv-PE38 fusion protein in E.coli

目的 :实现二硫键稳定的抗肝癌单链抗体与PE38融合 ,免疫毒素在大肠杆菌中的可溶性表达 ,为下游的活性检测等工作打下基础 .方法 :设计了两种不同的表达载体(GST、硫氧还蛋白促可溶表达载体 ) ,并通过改变宿主菌菌株、IPTG诱导浓度、诱导温度及诱导时间等 ,优化表达条件 ;通过ELISA法判断该免疫毒素中单链抗体的结合活性 .结果 :抗肝癌单链抗体与PE38融合蛋白在大肠杆菌Origami(DE3)的上清中得到表达 ,表达量占菌体总可溶蛋白量的2 1% ;ELISA法证实该融合蛋白保持了亲本抗体的特异性 .结论 :dsFv

AIM: To achieve the soluble expression of an immnotoxin consisting of an anti hepatoma disulfide stabilized scFv and truncated pseudomonas exotoxin (PE38). METHODS: dsFv PE38 fusion gene was inserted into two prokaryotic expression vectors, which contain the GST and Trx gene respectively, and was transformed into different host strains. The concentration of IPTG, time and temperature were adjusted and the specificity of the dsFv PE38 fusion protein was examined by ELISA. RESULTS: The fusion protein with M r 66 000 was successfully expressed in E.coli Origami (DE3) in soluble supernatant, which amounted to 21% of the total soluble proteins. ELISA showed that, like its parent disulfide stabilized scFv, dsFv PE38 had similar specificity to the hepatoma cells. CONCLUSION: The immunotoxin dsFv PE38 is successfully expressed in soluble supernatant, which will provide a new way to the study of soluble expression of other fusion proteins.