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单链抗体-碱性磷酸酶融合蛋白的制备 被引量:7

Production of single chain antibody-alkaline phosphatase fusion protein
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摘要 目的 :制备单链抗体 碱性磷酸酶融合蛋白。方法 :把碱性磷酸酶编码区插入pSTE2 8E5质粒DNA ,用scFvC6 6的重链和轻链可变区DNA取代scFv 8E5的重链和轻链可变区DNA ,构成表达载体pSTE2 C6 6 Ap在大肠杆菌中表达 ,用聚丙烯酰胺凝胶电泳和免疫印迹法分析产物活性。结果 :获得一个分子量为 75kD的scFvC6 6 Ap融合蛋白 ,可结合来自KG1a细胞裂解物中的一个分子量约 6 0kD的蛋白质带 ,其结合功能可通过其碱性磷酸酶活性直接测定。结论 :建立了一个用大肠杆菌制备单链抗体 碱性磷酸酶融合蛋白的方法 ,它可能取代常规的碱性磷酸酶标记方法而用于免疫检测。 Objective:Making single chain antibody(scFv)-alkaline phosphatase(Ap)fusion protein.Methods:An expression vector pSTE2-C66-Ap was constructed by sequentially inserting the Ap coding region into plasmid pSTE2-8E5 and replacing the VH-VL fragment of 8E5 by VH-VL fragment of C66.The fusion protein scFvC66-Ap was expressed in E.coli.and analysed by SDS-PAGE and immunoblotting.Results:Have obtained the scFvC66-Ap fusion protein with a molecular weight of 75 kD.It bound a 60 kD molecule from KG1a cell proteins on immunoblotting membrane detected directly by Ap enzymatic activity.Conclusion:A method permits the production of scFv-Ap conjugates in E.coli.which can replace conventionally prepared Ap-labeled antibodies in immunoassays.
出处 《中国免疫学杂志》 CAS CSCD 北大核心 2003年第10期704-706,共3页 Chinese Journal of Immunology
基金 国家自然科学基金资助 (批准号 3 0 170 3 49)
关键词 单链抗体 碱性磷酸酶 融合蛋白 Single chain antibody Alkaline phosphatase Fusion protein
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