抗人CD33单链抗体的基因构建、表达及其生物活性检测  

Construction,expression and functional characterization of single chain variable fragments (scFv) against human CD33 antigen

作　　者：陈小军[1] 王洋[1] 屈浩[1] 葛新顺[1] 左玉丰[1] 廖晓龙[1] 

机构地区：[1]中国医学科学院北京协和医学院(清华大学医学部)血液学研究所血液病医院免疫室,天津300020

出　　处：《细胞与分子免疫学杂志》2007年第12期1147-1149,1156,共4页Chinese Journal of Cellular and Molecular Immunology

摘　　要：目的:构建及表达抗人CD33单链抗体(抗CD33-scFv)基因,并检测其生物活性。方法:采用RT-PCR方法从分泌抗人类白细胞表面分化抗原CD33单克隆抗体(mAb)的杂交瘤细胞中克隆出VL和VH可变区基因,再通过重叠延伸拼接(splice-overlap extension)PCR方法在VH和VL可变区基因之间引入柔性连接肽(Gly4Ser)3,体外构建抗人CD33-scFv基因。将其克隆至原核表达载体PET-28a(+)并在大肠杆菌Rosetta(DE3)中表达。结果:SDS-PAGE和Westernblot分析结果表明,抗CD33-scFv在Rosetta(DE3)菌中获得高效表达,重组蛋白的相对分子质量(Mr)为30000,表达产物以不溶性包涵体形式存在,经过溶解包涵体,镍柱亲和层析纯化和体外复性过程,获得了高纯度的scFv片段。流式细胞术(FCM)分析结果证实抗CD33-scFv可与人类白细胞表面的分化抗原CD33结合,保留了鼠源性mAb的与CD33结合的活性。结论:重组抗人CD33-scFv基因构建与表达成功,并且通过复性得到有生物活性的scFv,为下一步针对髓系恶性肿瘤的靶向治疗奠定了基础。AIM： To construct and express the single chain variable fragments （scFv） gene against human CD33 antigen, and characterize its bioactivity. METHODS： The genes encoding the light and heavy chain variable regions were cloned by RT-PCR from a murine hybridoma cell line, which could produce monoclonal antibody （mAb） against human CD33 antigen. Then the light and heavy chain variable regions were fused together by a short peptide linker containing 1.5 amino acid （Gly4Ser） 3 using splice-overlap extensive PCR. The recombinant anti-CD33 scFv was subcloned into the expression vector pET28a （ ＋ ） and expressed in E. coil Rosetta after induction by IPTG. RESULTS： SDS-PAGE and Western blot analysis showed that the recombinant anti-CD33 scFv gene was expressed in the form of inclusion body in E. coil Rosetta, and the purified fusion protein was obtained after a series of purification steps including cell lysis, inclusion body solubilization, Ni^2＋ metal affinity chromatography and protein refolding. Flow cytometry（FCM） analysis showed that the scFv could react with human CD33 antigen. CONCLUSION： Recombinant anti-CD33 scFv gene has been successfully constructed and expressed in E. coil Rosetta, which could provide foundation for the future target therapy to the myeloid leukemia.

关 键 词：CD33 SCFV 原核表达 复性 

分 类 号：R392.11[医药卫生—免疫学]