抗慢性粒细胞白血病细胞人源化单链抗体的原核表达及其活性

Prokaryotic expression and activity of humanized single-chain variable fragment against chronic myelogenous leukemia cells

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 single-chain variable fragment（hscFv） against chronic myeloid leukemia （CML） cells in prokaryotic cells and determine its antigen-binding activity. Methods The hscFv gene was amplified from recombinant plas- mid pUC57-hscFv and inserted into prokaryotic expression vector pET-32a（＋） with a 6 × His tag. The constructed recombinant plas- mid pET-32a-hscFv was transformed to E. coli BL21 （DE3） for expression under induction of IPTG. The expressed fusion protein was purified by Ni2^2＋-NTA chromatography, then analyzed for purity by SDS-PAGE, for reactogenicity by Western blot, and for antigen- binding activity by indirect IFA. Results PCR, restriction analysis and sequencing proved that recombinant plasmid pET-32a-hscFv was constructed correctly. The expressed recombinant fusion protein, with a relative molecular mass of about 46 000, contained about 37% of total somatic protein and mainly existed in a soluble form, which reached a purity of 94% after purification and showed specific binding to mouse monoclonal antibody against 6×His tag and the antigen on surface of K562 cells. Conclusion The hscFv against CML cells was successfully expressed in prokaryotic cells and purified, which laid a foundation of further application of hscFv for clinical molecular diagnosis and target biotherapy of CML.