抗人精浆蛋白单克隆抗体Fab段基因的克隆及其在大肠杆菌的表达  

Cloning and expression in E.coli of Fab gene of an anti-human γ seminoprotein monoclonal antibody

作　　者：杨洁[1] 郝晓柯[2] 孙脊峰[1] 赵晶[3] 于翠娟[3] 许彦鸣[3] 金明[3] 王成济[3] 杨安钢[3] 

机构地区：[1]第四军医大学唐都医院肾脏内科,陕西西安710038 [2]第四军医大学西京医院临床分子生物学研究中心,陕西西安710032 [3]第四军医大学生物化学与分子生物学教研室,陕西西安710032

出　　处：《免疫学杂志》2004年第4期310-312,316,共4页Immunological Journal

基　　金：国家高技术研究发展计划"863"项目 (2 0 0 1AA2 1 532 1 );教育部高等学校骨干教师资助计划项目 (2 0 0 0 65 66);国家杰出青年科学基金项目 (3992 50 36)资助

摘　　要：目的　获得鼠抗人精浆蛋白单克隆抗体Fab段基因并在大肠杆菌中表达。方法　从分泌抗人精浆蛋白单克隆抗体的杂交瘤细胞系E4B7中提取总RNA ,用逆转录聚合酶链反应技术 (RT PCR)克隆Fd段和κ链基因 ;构建到噬粒pComb3中 ;并用ELISA、Westernblotting和免疫细胞化学分析证明表达的Fab段特异性结合人精浆蛋白。结果　从分泌抗人精浆蛋白的鼠单克隆抗体杂交瘤细胞系E4B7中克隆出了重链Fd段和κ链基因 ,经序列测定表明 ,Vκ 属于鼠免疫球蛋白ⅩⅢ家族 ,VH与鼠免疫球蛋白MUSIGHAEI同源性最高。将该Fd和κ链基因克隆到表达载体pComb3中 ,在大肠杆菌中获得了表达。结论　ELISA、Westernblotting和免疫细胞化学分析表明 。Objective Fab gene from anti-human γ seminoprotein monoclonal antibody was cloned and expressed in E.coli. Me- thods Total RNA was extracted from the hybridoma cell line E 4B 7 secreating anti-human γ seminoprotein monoclonal antibody, and subjected to reverse transcription. The Fd and κ gene were amplified by PCR, and then cloned into the expressing vector pComb3 and expressed in E.coli. The antibody fragment activity to γ seminoprotein was identified by ELISA, western blot, and immunocytochemistry. Results The Fd and κ gene were cloned. Sequencing analysis showed that the V κ share a close homology with mouse Ig kappa family ⅩⅢ and V H share a close homology with mouse Ig MUSIGHAEI. The Fd and κ gene were cloned into the expressing vector pComb3 and expressed in E.coli. Conclu- sion ELISA,Western blotting, and immunocytochemistry analysis demonstrated that bacterial expressed Fab fragment specifically bound to human-γ seminoprotein.

关 键 词：人精浆蛋白 单克隆抗体 FAB段 基因表达 

分 类 号：R392.11[医药卫生—免疫学]