【摘要】 目的从全人源噬菌体抗体库中筛选与人骨肉瘤细胞MG63特异性结合的单链抗体(scFv)并进行表达、纯化及鉴定。方法采用噬菌体表面展示技术,以正常人成骨细胞系hFOBl.19为阴性筛选细胞,人骨肉瘤细胞系MG63为阳性筛选细胞,经过"吸附-洗脱-扩增"过程筛选并富集特异性抗体,ELISA检测并进行PCR扩增及测序鉴定。获得的阳性噬菌体感染大肠杆菌HB2151,IPTG诱导后经镍亲和层析柱纯化,用SDS-PAGE鉴定表达、纯化效果,ELISA检测可溶性特异性单链抗体的亲和力、特异性及稳定性。MTT法检测纯化后的单链抗体对骨肉瘤细胞MG63增殖的影响。结果通过筛选、PCR扩增和序列分析确定scFv4的片段包含免疫球蛋白序列,其表达产物可与抗原结合;纯化后的单链抗体对骨肉瘤细胞MG63增殖有明显的抑制作用,且其抑瘤作用与质量浓度成正相关。未加蛋白保护剂的纯化抗体4℃下其抗体活性可保持约5周,添加蛋白保护剂的纯化抗体则可在4℃下保持6~7周。结论利用噬菌体表面展示技术成功筛选出能与人骨肉瘤细胞MG63特异性结合的scFv,在大肠杆菌中成功表达并获得基因序列,抗体能抑制MG63细胞的增殖且稳定性良好。更多还原

【Abstract】 Osteosarcoma is a most common primary malignant bone tumor. In this paper we tried to identify specific human single-chain Fv genes against osteosarcoma cell MG63 from a human scFv phage library followed negative panning by Hfob1.19 and positive panning by osteosarcoma cell line MG63 successively. The positive clone was amplified by PCR, and then sequenced; the soluble scFv product was expressed in E. coli and analyzed by SDS-PAGE and ELISA. Effects of the scFv product on MG63 cell proliferation were detected by MTT method.Data showed scFv4 gene fragment was successfully expressed in E. coli, appearing 36 kDa on SDS-PAGE gel. After purification by Ni-NTA affinity chromatographic column, the scFv4 product could recognize MG63 cell and suppressed the growth of MG63 cell in a dose-dependent manner. In addition, the stability analysis revealed that the binding activity of scFv4 remained in 5 weeks without any treatment at 4 ℃, and the storage life extended to 6-7 weeks with presence of OVA and PMSF at 4 ℃. In summary, scFv4 specific to MG63 cells has been identified,which will set a basis for further research work on carcinogenesis and targeted therapy of osteosarcoma.更多还原