抗淋巴样增强因子-1羧基端单克隆抗体的制备及其表位的初步鉴定  

Preparation and antigenic epitope identification of the monoclonal antibody against lymphoid enhancer factor-1 carboxyl terminus

作　　者：杨明珍[1] 李鹏[1] 张竹君[1] 易维京[1] 李淑慧[1] 赵利军[2] 胡川闽[1] 

机构地区：[1]第三军医大学医学检验系临床生物化学教研室,重庆400038 [2]美国北卡罗来纳大学化学与生物化学系

出　　处：《免疫学杂志》2008年第2期142-145,共4页Immunological Journal

基　　金：国家自然科学基金面上项目(30570373)

摘　　要：目的制备抗淋巴样增强因子-1(LEF-1)羧基端86个氨基酸(Lct86)特异性单克隆抗体,并初步明确其表位,为进一步研究LEF-1C端特异序列的功能奠定基础。方法从Jurkat细胞株中扩增LEF-1C端特异序列cDNA,并克隆入PQE40、pET32a原核表达载体;分别转化入M15、BL21(DE3)感受态菌,IPTG诱导表达;纯化PQE40-Lct86融合蛋白作为免疫原免疫Balb/c小鼠制备单克隆抗体,以pET32a-Lct86融合蛋白鉴定单克隆抗体特异性。LEF-1C端特异序列不同长度的基因片段定向克隆入PQE40,鉴定各片段的表达,利用Western blotting初步鉴定单克隆抗体的表位。结果成功构建了PQE40-Lct86、pET32a-Lct86重组原核表达载体;纯化获得PQE40-Lct86融合蛋白;得到一株特异性的抗LEF-1C端单克隆抗体TMAb1,证实其表位位于437aa^454aa。结论成功制备了一株特异性的抗LEF-1C端单克隆抗体TMAb1,并鉴定其抗原识别表位位于437aa^454aa,为进一步研究LEF-1C端的功能奠定基础。Objective To prepare monoclonal antibody against LEF-1 carboxyl terminus 86 amino acids （Lct86）, and identify the antigenic epitope recognized by this monoclonal antibody. Methods LEF-1 carboxyl terminus cDNA was amplified from Jurkat cell line, and then cloned into expression vector pE-332a and PQE40. The recombinants were transformed into BI21 （DE3） and M15 respectively, and then induced with IPTG. Balb/c mice were immunized with purified PQF40-Lct86 and the specificity of monoclonal antibody was tested with pE332a-Lct86. Furthermore, different length gene fragments of LEF-1 carboxyl terminus were subcloned in-frame into PQE40. These construct plasmids were transformed into M15 and induced to expression, and then were used to identify antigenic epitope of LEF-1 carboxyl terminus recognized by the monoclonal antibody. Results PQF40-Lct86 and pE332a-Lct86 recombinant plasmids were constructed. After purification, we obtained purified PQE40-Lct86 and had one specific monoclonal antibody against LEF-1 carboxyl terminus（TMAb1 ）, which could recognize the antigenic epitope （437aa- 454aa）. Conclusion One specific monoclonal antibody （TMAb1） which can recognize antigenic epitope （437aa- 454aa） of the LEF-1 carboxyl terminus is successfully prepared and would be useful to explore the biological role of the LEF-1 carboxyl terminus.

关 键 词：LEF-1 单克隆抗体 表位 

分 类 号：R392[医药卫生—免疫学]