抗人精浆蛋白单链抗体基因在HeLa细胞中的表达和活性鉴定

目的　在HeLa细胞中表达抗人γ 精浆蛋白 (γ Sm)单链抗体 (scFv)基因 ,并进行亲和活性测定。方法　在已经构建抗人γ SmscFv基因基础上 ,将基因克隆入真核分泌表达载体 pSecTag2 B中 ,并转染HeLa细胞进行表达。表达产物纯化后 ,用流式细胞仪进行亲和活性测定。结果　表达产物经SDS PAGE和Western印迹实验证实为约30ku的特异蛋白条带 ,纯化后经流式细胞仪检测可以特异性地结合PC 3细胞。结论　获得了可与PC 3细胞特异结合的抗人γ SmscFv ,为进一步临床应用奠定基础

Expression of single chain Fv antibody gene specific for human γ-seminoprotein in HeLa cells and identification of binding affinity

Objective To express anti-human γ-seminoprotein (γ-Sm) single chain Fv antibody (scFv) gene in HeLa cells and identify its binding affinity for PC-3 cells. Methods The anti-human γ-Sm scFv was cloned into the pSecTag2-B expression plasmid. Then the pSecTag2-B plasmids containing the anti-human γ-Sm scFv gene were transfected by HeLa cells. The expression products were analyzed by both SDS-PAGE and Western blot, then were purified with Ni 2+-NTA superflow affinity chromatography. The binding affinity for PC-3 cells was measured by flow cytometry. Results The expression products of the anti-human γ-Sm scFv, whose relative molecular mass (Mr) was about 30 000, were confirmed by SDS-PAGE and Western blot. After being purified with Ni 2+-NTA superflow affinity chromatography, the anti-human γ-Sm scFv showed significantly strong binding with PC-3 cells. Conclusion The anti-human γ-Sm scFv which can bind with PC-3 cells has been successfully obtained for the potential use in clinical studies.