抗人CD44单链抗体基因的构建、表达及活性测定

Construction and Expression of Single Chain Variable Fragments(ScFv)Against Human CD44 Antigen

目的:构建表达抗CD44单链抗体(ScFv),并进行体外活性的测定。方法:采用RT-PCR方法从分泌鼠抗人CD44mAb的杂交瘤细胞中克隆出VH和VL可变区基因,再通过重叠延伸拼接PCR方法在VH和VL。可变区基因之间引入连接肽(G4S)3,体外构建抗人CD44单链抗体基因。将其克隆至表达载体PET22b并在大肠杆菌中表达。SDS-PAGE和Flag-FITC分析结果表明,抗CD44-ScFv在BL21菌中获得表达,表达产物以可溶性蛋白形式存在,用镍柱亲和层析纯化和体外复性过程,获得了高纯度的单链抗体片段。结果:证实抗CD44-ScFv可与人类白细胞表面的分化抗原CD44结合,保留了鼠源性单抗与CD44结合活性。结论:抗人CD44-ScFv的构建与表达,为下一步针对髓系白血病的靶向治疗奠定了基础。

Obiective To construct antibody express anti-CD44 ScFv,And to determine reaction in vitro . Method The genes encoding for the light and heavy chain variable regions were cloned by RT-PCR from a murine monoclonal hybridoma cell line,which could produce monoclonal antibody to recognize CD44 antigen.Then fused the light and heavy chain variable regions together by a short peptide linker containing 15 amino acid(G1y4Set)3 using splice-overlap extensive PCR.The recombinant anti-CD44-ScFv was subcloned into the expression vector pET22b and induced to be expressed by IPTG in E.coli BL21.SDS-PAGE and Flag-FITC showed that the recombinant anti-CD44-ScFv gene was expressed in E. coli BL21.ScFv expression was in the form of an inclusion bodies and the purified fusion protein was obtained after a series of purification steps ,Ni metal affinity chromatography and protein refolding.Rusule:: the ScFv can react with human CD44 antigen.In conclusion, recombinant anti-CD44-ScFv gene has been Successful constructed and expressed in E.coli B21.Conclusion: which could provide a basic study for the future target therapy to the B lymphoid leukemia and B lymphoma.