抗人CD33单克隆抗体(HIM3-4、WM53、M195)抗原识别表位的初步研究

Preliminary study of antigenic epitopes recognized by mAb(HIM3-4,WM53,M195) against human CD33

目的：确定抗人CD33单克隆抗体（HIM3-4、WM53、M195）抗原识别表位所在区域，进而阐明抗体识别表位不同于其介导的生物学作用之间的关系。方法：将四个CD33胞外区不同长度的基因片段定向克隆入真核表达载体pDisplay，构建成真核表达载体pDisplay／EP1、2,3、4，分别将空载体和构建的质粒通过磷酸钙沉淀法转染入293T细胞，瞬时转染48小时后利用流式细胞仪通过抗血凝素A（HA）抗体标记以检测各质粒在细胞表面的表达，同时检测HIM3-4、WM53、M195和不同区段的结合活性。结果：抗人CD33单克隆抗体HIM3-4能识别pDisplay／EP1、2、3表达的蛋白，WM53能识别pOisplay／EP1表达的蛋白，M195则均能识别四个片段所表达的蛋白。结论：HIM3-4、WM53、M195识别的CD33抗原表位分别位于115—170、17—60、170—262氨基酸。

Objective：To identify the regions of antigenic epitopes recognized by monoclonal antibodies （mAb） （HIM3-4, WM53 ,M195） against human CD33. Methods：Four different length gene fragments of human CD33 extracellular region were cloned in frame into the mammalian expression vector pDisplay. Then these constructed and control plasmids were transfected into the 293T cells with calcium phosphate-DNA coprecipitation. Forty-eight hours after transfection, the efficiency of transfection was analyzed by FACS with the anti-HA（ Hemagglutinin A） mAb. The regions of CD33 antigen recognized by anti-CD33 antibodies （ HIM3-4, WM53, M195） were analyzed by FACS respectively. Results：The proteins expressed by pDisplay/EP1,2 and 3 were recognized by mAb HIM3-4. The proteins expressed by pDisplay/EP1 were recognized by mAb WM53. The proteins expressed by pDisplay/EP1,2, 3 and 4 were recognized by mAb M 195. Conclusion：The antigenic epitopes recognized by HIM3-4 are located in the amino acid residue （AAR） 115-170 of CD33 antigen. The antigenic epitopes recognized by WM53 are located in the AAR 17-60 of CD33 antigen. The antigenic epitopes recognized by M195 are located in the AAR 170-262 of CD33 antigen.