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目的原核表达抗慢性粒细胞白血病(Chronic myelogenous leukemia,CML)细胞人源化单链抗体(Humanized sin-gle-chain variable fragment,hscFv),并检测其抗原结合活性。方法从重组质粒pUC57-hscFv中扩增hscFv基因,插入含6×His标签的原核表达载体pET-32a(+)中,构建重组表达质粒pET-32a-hscFv,转化E.coli BL21(DE3),IPTG诱导表达。表达的重组融合蛋白经Ni2+-NTA亲和层析纯化后,SDS-PAGE分析其纯度;Western blot分析其反应原性;间接免疫荧光试验检测其抗原结合活性。结果重组表达质粒经PCR、双酶切及测序鉴定证明构建正确;表达的重组融合蛋白相对分子质量约为46 000,表达量约占菌体总蛋白的37%,主要以可溶性形式存在;纯化的重组融合蛋白纯度为94%,可与鼠抗6×His单抗及K562细胞表面抗原特异性结合。结论成功原核表达并纯化了抗CML细胞hscFv,为其进一步应用于CML的临床分子诊断和生物靶向治疗奠定了基础。
Objective To express humanized sin-gle-chain variable fragment (hscFv) in Chronic myelogenous leukemia (CML) cells and detect its antigen-binding activity. Methods The hscFv gene was amplified from the recombinant plasmid pUC57-hscFv and inserted into the prokaryotic expression vector pET-32a (+) containing 6 × His tag. The recombinant plasmid pET-32a-hscFv was constructed and transformed into E.coli BL21 (DE3) IPTG induced expression. The recombinant fusion protein was purified by Ni2 + -NTA affinity chromatography and its purity was analyzed by SDS-PAGE. The antigenicity was determined by Western blot. The antigen-binding activity was detected by indirect immunofluorescence assay. Results The recombinant plasmid was confirmed by PCR, double enzyme digestion and sequencing. The recombinant fusion protein was about 46 000 in molecular weight and accounted for about 37% of the total bacterial protein. The recombinant fusion protein mainly existed in soluble form. The purified The purity of recombinant fusion protein was 94%, which could specifically bind to mouse anti-6 × His monoclonal antibody and K562 cell surface antigen. Conclusion The prokaryotic expression and purification of hscFv against CML cells have laid the foundation for further clinical molecular diagnosis and bio-targeted therapy of CML.