低免疫原性的新型葡激酶ΔNMSak在大肠杆菌中的高效表达和分离纯化

为了降低野生型葡激酶 (wild- type staphylokinase,wt- Sak)的免疫原性 ,对已构建的葡激酶N端缺失突变体 (ΔNSak) c DNA进行改造 ,将其主要的抗原决定簇编码序列突变为丙氨酸密码子 .该突变体 (ΔNMSak) c DNA与原核表达载体 p LY- 4重组后 ,转化大肠杆菌 JF1 1 2 5.经温度诱导 ,ΔNMSak获得高效表达 ,重组蛋白占全菌总蛋白的 60 % ,以包涵体形式存在 .包涵体经洗涤 ,8mol/L尿素溶解 ,稀释复性 ,离子交换色谱一步分离至电泳纯 ,纯度达 95%以上 ,分子量与理论值相符 ,比活性 8.5× 1 0 4 HU/mg.经 ELISA法、发色底物法测定 ,ΔNMSak与 wt- Sak制备的兔抗wt- Sak抗血清的免疫反应性显著降低 ,经抗血清温育后 ,wt- Sak活性下降程度远高于 ΔNMSak.ΔNMSak、wt- Sak分别免疫豚鼠 ,以 ELISA法测定豚鼠血清中相应抗体的效价 ,ΔNMSak组的抗体效价明显低于 wt- Sak组 ,表明 ΔNMSak的免疫原性显著下降 .

Construction,Over-expression in E. coli and Purification of a Novel Variant of Staphylokinase with Reduced Immunogenicity *

In order to reduce the immunogenicity of staphylokinase (Sak),cDNA of a novel variant(ΔNMSak) was constructed by substitution mutagenesis of an immunodominant epitope with alanine,confirmed by nucleotide sequencing.The mutant cDNA was ligated with prokaryotic expression vector pLY 4 and transformed into E.coli JF1125.After temperature induction,over 60% expression level of ΔNMSak was achieved.ΔNMSak was isolated and purified by washing and solubilization of inclusion body,renaturation and ion exchange chromatography.The final product displayed a single band with a corresponding molecular weight 14 kD in SDS PAGE.The purity was over 95% and the specific activity of ΔNMSak was 8.5×10 4 HU/mg.The immunoreactivity of ΔNMSak to polyclonal antibody against wt Sak was sharply reduced to a very low level determined by both chromogenic substrate assay and ELISA.Antiserum titres raised against ΔNMSak in guinea pigs were much lower than those of wt Sak,determined by ELISA.It provided the basis for further reconstruction of wt Sak by protein engineering.