摘要
目的 :建立简便快速的单链抗体检测方法。方法 :用PCR法和DNA重组技术构建了突变型碱性磷酸酶基因和单链抗体 突变型碱性磷酸酶融合基因 ;利用IPTG诱导融合基因的表达 ,并用pNPP底物液检测碱性磷酸酶的活性 ,用ELISA法检测和比较了不同单链抗体的相对活性。结果 :实现重组突变型碱性磷酸酶基因在大肠杆菌中的功能性表达 ,且活性较重组野生型碱性磷酸酶活性高 30倍左右。重组单链抗体 突变型碱性磷酸酶融合基因在大肠杆菌中获得功能性表达 ,周质中表达产物具有双功能 ,并成功地利用该系统分析了不同单链抗体的相对活性。结论 :该单链抗体
Objective:To establish a convenient ScFv alkaline phosphatase system for detecting the bioactivity of ScFv. Methods:By using PCR and DNA recombination methods,the mutative alkaline phosphatase (Apm) gene and ScFv Apm fused gene were constructed.After the recombinant proteins were expressed by the inducement of IPTG,the bioactivity of alkaline phosphatase was analyzed with pNPP and the bioactivity of three different ScFv was analyzed with ELISA.Results:The mutative alkaline phosphatase was functionally expressed in E.coli ,and its bioactivity was about 30 times higher than that of intact alkaline phosphatase. The ScFv Apm fusion protein was secreted into the periplasm of E.coli ,and the fusion protein retained the activity of two moieties.This detection system was successfully used to analyze the relative affinity of three different ScFv.Conclusion:The ScFv alkaline phosphatase system is effective in analyzing the bioactivity of ScFv.
出处
《军事医学科学院院刊》
CSCD
北大核心
2001年第4期286-289,共4页
Bulletin of the Academy of Military Medical Sciences
基金
国家高技术研究和发展计划项目 ( 10 2 0 9 0 3 0 1)
