抗人CD19单链抗体基因的构建、表达及功能测定

采用RT-PCR方法从分泌抗人类白细胞表面分化抗原CD19单克隆抗体的杂交瘤细胞中克隆出V_H和V_L可变区基因，再通过重叠延伸拼接（spliceoverlap extension）PCR方法在V_H和V_L可变区基因之间引入连接肽(Gly4Ser)3,体外构建抗人CD19单链抗体（抗CD19-ScFv）基因。将其克隆至表达载体PET28a并在大肠杆菌中表达。SDS-PAGE和Westernblot分析结果表明，抗CD19-ScFv在BL21（DE3）菌中获得表达，重组蛋白的相对分子量为27kD，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，获得了高纯度的单链抗体片段。流式细胞分析结果证实抗CD19ScFv可与人类白细胞表面的分化抗原CD19结合，保留了鼠源性单抗与CD19结合活性。抗人CD19-ScFv的构建与表达，为下一步针对B淋巴系统恶性肿瘤的靶向治疗奠定了基础。

Construction and Expression of Single Chain Variable Fragments (ScFv) Against Human CD19 Antigen

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 CD19 antigen on human B lymphocyte. Then fused the light and heavy chain variable regions together by a short peptide linker containing 15 amino acid (Gly4Ser)3 using splice-overlap extensive PCR. The recombinant anti-CD19- ScFv was subcloned into the expression vector pET28a and induced to be expressed by IPTG in E.coli BL21. SDS-PAGE and Western blot analysis showed that the recombinant anti-CD19-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 including cell break , inclusion body solubilization, Ni ~2+ metal affinity chromatography and protein refolding. Flow cytometry analysis showed that the ScFv can react with human CD19 antigen. In conclusion, recombinant anti-CD19-ScFv gene has been successful constructed and expressed in E.coli BL21, which could provide a basic study for the future target therapy to the B lymphoid leukemia and B lymphoma.