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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 2个靶抗原结合的活性
OBJECTIVE: To construct and express anti-CD3 / anti-CD20 micro-bifunctional antibody and to determine the biological activity of the micro-bifunctional antibody. Methods: The anti-CD3 / anti-CD20 micro-bifunctional antibody was constructed by PCR and overlapPCR. The DNA sequence was determined by dideoxy termination method. The product was purified by affinity chromatography and the purified product was identified by Western blot and size exclusion chromatography. FACS and rosette assays were used to identify the binding activity of the purified product to target cells. Results: The results of DNA sequencing showed that the anti-CD3 / anti-CD20 micro-bifunctional antibody was successfully constructed, the soluble product yield reached 1mg / ml and the purified product dimer ratio reached 90%, which was consistent with Jurkat (CD3 + ) And Daudi cells (CD20 +), and can simultaneously bind to Jurkat and Daudi cells to form a rosette. CONCLUSION: The anti-CD3 / anti-CD20 micro-bifunctional antibody was successfully constructed for the first time using the Diabody format and was highly expressed. The expressed product had the activity of binding to the corresponding two target antigens