天然抗角蛋白单克隆抗体3B4的噬菌体呈现的scFv的构建及表达  

Construction and expression of scFv phage antibody from natural anti-keratin monoclonal antibody 3B4

作　　者：管海宏[1] 付萌[1] 李巍[1] 夏汝山[1] 王刚[1] 刘玉峰[1] 

机构地区：[1]第四军医大学西京医院皮肤科,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2005年第4期470-472,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金重点项目(No.30330510);全军医药卫生科研基金资助项目(No.01Z086)

摘　　要：目的:构建并表达噬菌体呈现的天然抗角蛋白单克隆抗体(mAb)3B4的单链抗体(scFv),并测定其活性。方法:分别以pMD18TmAb3B4VH和pMD18TmAb3B4VL为模板进行PCR,扩增mAb3B4的VH和VL基因,然后将其依次插入噬菌体表达载体pscMH中。经DNA序列测定正确后,转化大肠杆菌,用辅助噬菌体VCSM13超感染诱导表达scFv;用ELISA检测其与亲本mAb3B4的抗原结合活性的改变。结果:对扩增的mAb3B4的VH和VL基因进行测序,结果表明VH基因与原序列完全一致,VL基因在骨架区FR1有1个碱基发生突变。用VCSM13超感染后能检测到scFv的表达,其与亲本一样具有多反应性,但抗原结合模式不完全相同。结论:成功地构建并表达了天然抗角蛋白mAb3B4噬菌体呈现的单链抗体,表达的scFv与mAb3B4的抗原结合活性有一定的差异,为探讨天然自身抗体的抗原结合模式奠定了基础。AIM: To construct and express the scFv phage antibody from natural anti-keratin monoclonal antibody 3B4 in E.coli and then compare its antigenic specificity with that of the parental antibody 3B4. METHODS: The V_H and V_L of 3B4 were amplified by PCR from pMD-18T- mAb 3B4 V_H and pMD-18T- mAb 3B4 V_L, and then cloned into the phagemid pscMH. E.coli was transfected by recombined pscMH and superinfected by the helper phage VCSM13. Then the antigenic specificity of the scFv concentrated from the supernatant of the transfected E.coli was detected by indirect ELISA. RESULTS: DNA sequence analysis showed that the scFv sequence was the same as the original sequence except that there is one base mutation in the FR1 of V_L. Indirect ELISA showed that the scFv phage antibody was polyreactive as mAb 3B4, but its antigenic specificity was not the same with that of mAb 3B4. CONCLUSION: The scFv phage antibody from natural anti-keratin monoclonal antibody 3B4 was successfully constructed and expressed. Its antigenic specificity was different from its parental antibody 3B4.

关 键 词：天然抗角蛋白自身抗体 SCFV 多反应性 

分 类 号：R392.11[医药卫生—免疫学]