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.
出处
《免疫学杂志》
CAS
CSCD
北大核心
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
关键词
人精浆蛋白
单克隆抗体
FAB段
基因表达
Human-γ seminoprotein
Monoclonal antibody
Fab fragment
Gene expression
