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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 碱性磷酸酶检测系统能够方便地应用于单链抗体的活性分析
Objective: To establish a simple and rapid single-chain antibody detection method. Methods: Mutant alkaline phosphatase gene and single-chain antibody mutant alkaline phosphatase fusion gene were constructed by PCR and DNA recombination technology. The expression of fusion gene was induced by IPTG, and the alkaline phosphatase Activity, the relative activity of different single-chain antibodies was detected and compared by ELISA. Results: The recombinant mutant alkaline phosphatase gene was expressed in Escherichia coli and the activity of wild-type alkaline phosphatase was 30-fold higher than that of the wild-type alkaline phosphatase. The recombinant single chain antibody mutant Alkaline Phosphatase fusion gene was expressed in Escherichia coli, and the expressed product in periplasm was bifunctional. The relative activity of different single chain antibodies was successfully analyzed by this system. Conclusion: The single-chain antibody alkaline phosphatase detection system can be conveniently applied to the activity analysis of single chain antibody