Chen Xiao-Jun, Wang Yang, Qu Hao, Ge Xin-Shun, Zuo Yu-Feng, Liao Xiao-Long
Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Dec;23(12):1147-9.
To construct and express the single chain variable fragments (scFv) gene against human CD33 antigen, and characterize its bioactivity.
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 15 amino acid (Gly(4)Ser)(3) using splice-overlap extensive PCR. The recombinant anti-CD33 scFv was subcloned into the expression vector pET28a(+) and expressed in E.coli Rosetta after induction by IPTG.
SDS-PAGE and Western blot analysis showed that the recombinant anti-CD33 scFv gene was expressed in the form of inclusion body in E.coli 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.
Recombinant anti-CD33 scFv gene has been successfully constructed and expressed in E.coli Rosetta, which could provide foundation for the future target therapy to the myeloid leukemia.
构建并表达抗人CD33抗原的单链可变片段（scFv）基因，并对其生物活性进行鉴定。
通过RT-PCR从能产生抗人CD33抗原单克隆抗体（mAb）的鼠杂交瘤细胞系中克隆编码轻链和重链可变区的基因。然后使用含15个氨基酸（Gly(4)Ser）(3)的短肽接头通过重叠延伸PCR将轻链和重链可变区融合在一起。将重组抗CD33 scFv亚克隆到表达载体pET28a(+)中，并在IPTG诱导后在大肠杆菌Rosetta中表达。
SDS-PAGE和Western blot分析表明重组抗CD33 scFv基因在大肠杆菌Rosetta中以包涵体形式表达，经过细胞裂解、包涵体溶解、Ni(2+)金属亲和层析和蛋白质复性等一系列纯化步骤后获得了纯化的融合蛋白。流式细胞术（FCM）分析表明scFv能与人CD33抗原反应。
重组抗CD33 scFv基因已成功构建并在大肠杆菌Rosetta中表达，可为未来髓系白血病的靶向治疗提供基础。
