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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。
OBJECTIVE: To screen, purify and identify single chain antibody (scFv) that specifically binds to human osteosarcoma cell MG63 from whole human phage antibody library. Methods The phage display technique was used to screen human osteosarcoma cell lines hFOBl.19 as negative cells. The human osteosarcoma cell line MG63 was screened for positive cells and screened and enriched by the process of “adsorption - elution - amplification” Antibodies, ELISA detection and PCR amplification and sequencing identification. The positive phage were infected with E.coli HB2151. After induced by IPTG, the recombinant protein was purified by nickel affinity chromatography. The expression and purification of the recombinant protein were confirmed by SDS-PAGE. The affinity, specificity and stability of the soluble specific single-chain antibody were detected by ELISA. Effect of purified single chain antibody on proliferation of osteosarcoma MG63 cells by MTT assay. Results The fragment of scFv4 was confirmed to contain immunoglobulin sequence by screening, PCR amplification and sequence analysis. The expressed product was able to bind to the antigen. The purified single chain antibody could significantly inhibit the proliferation of osteosarcoma MG63 cells. The effect is positively correlated with the mass concentration. Purified antibodies without protein protectors maintained their antibody activity at 4 ° C for about 5 weeks and purified antibodies with protein protectors maintained at 4 ° C for 6 to 7 weeks. Conclusion The scFv, which can specifically bind to human osteosarcoma cell MG63, was successfully screened by phage display technology and successfully expressed in Escherichia coli. The antibody can inhibit the proliferation of MG63 cells and has good stability.