微型双功能抗体抗CD3/抗CD20的构建和表达

Study on the construction and expression of anti-CD3/anti-CD20 Diabody

目的 :构建和表达抗CD3/抗CD2 0微型双功能抗体 ,并测定该微型双功能抗体的生物学活性。方法 :采用PCR和overlapPCR方法构建抗CD3/抗CD2 0微型双功能抗体 ,并用双脱氧终止法测定DNA序列 ;采用亲和层析法纯化该产物 ,并用Westernblot和分子排阻层析鉴定纯化产物 ;采用FACS法和玫瑰花环试验鉴定纯化产物与靶细胞的结合活性。结果 :DNA序列测定结果表明 :抗CD3/抗CD2 0微型双功能抗体已构建成功 ,表达可溶性产物的产量达 1mg/ml以上 ,纯化产物中二聚体的比例达 90 % ,具有与Jurkat(CD3+ )和Daudi细胞 (CD2 0 + )结合的活性 ,且能同时与Jurkat和Daudi细胞结合形成玫瑰花环。结论 :利用Diabody形式 ,首次成功地构建了抗CD3/抗CD2 0微型双功能抗体 ,并获得较高表达 ,表达产物具有与相应

Objective:To construct and express anti CD3/anti CD20 Diabody and identify its biological activity.Methods:PCR and overlap PCR were used to construct anti CD3/anti CD20 Diabody.DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide.The product was purified by affinity chromatography and analyzed by both the detection of Western blot and size exclusion chromatography;its antigen binding activity was examined by FACS and rosetting assay.Results:The data of DNA sequence showed that the anti CD3/anti CD20 Diabody was corrected.The Diabody was recovered in high yield(up to 1 mg/ml) after E taq purification and predominantly(90%) as a dimer.The Diabody binded Jurkat cells(CD3 +) and Daudi cells (CD20 +),respectively.Furthermore,the Diabody was capable of simultaneous binding to Jurkat cells and Daudi cells as shown by cellular rosetting.Conclusion:The anti CD3/anti CD20 BsF(ab') 2 was first recast into the Diabody format and succeeded to obtain high level expression.The results of some biological activity experiments indicated that the Diabody could bind to Jurkat cells and Daudi cells.