人survivin蛋白的表达、纯化与单克隆抗体制备

Prokaryotic expression of human survivin and preparation of anti-survivin monoclonal antibody

目的:表达人survivin重组蛋白并制备单克隆抗体。方法:表达含有survivin基因的重组质粒pRSETB-survivin,表达人survivin重组蛋白,以此为抗原,常规方法免疫BALB/c小鼠,取其脾细胞与NS21细胞融合,得稳定分泌survivinmAb的杂交瘤细胞株,ELISA检测mAb的相对亲和力,Westernblot检测mAb的特异性。间接免疫荧光观察骨肉瘤细胞中survivin的表达情况。结果:重组载体pRSETB-survivin转化入大肠杆菌BL21中表达所得蛋白经Westernblot验证为目的蛋白。筛选出2株能稳定分泌特异性抗人survivin的mAb杂交瘤细胞株,免疫球蛋白亚类均为IgG类;单克隆抗体的亲和常数为S11.52×10-9mol/L,S22.31×10-9mol/L。Westernblot结果显示,2株单抗识别相对分子质量为20000的survivin单一条带,并发现在骨肉瘤细胞中survivin主要表达在胞浆内。结论:成功地表达出人survivin重组蛋白,制备出抗人survivinmAb,为进一步用于survivin的免疫学检测及骨肉瘤生物学治疗奠定了基础。

Objective To express human survivin protein in E. coli. and to prepare monoclonal antibody against it. Mothods The recombinant plasmid pRESTB-survivin was transformed into E. coli. BL21, and then expressed survivin. Recombinant human survivin protein was used as antigen to immunize BALB/c mice. Monoclonal antibodies against survivin were prepared by normal hybridoma technology. The relative affinity of mAbs was determined by ELISA. Specificity of mAbs was analyzed by Western blot. Expression of survivin in osteosarcoma cell line MG62 was investigated by immunofluorescence microscopy. Results SDS-PAGE showed the expression of recombinant protein of human survivin. Two hybridmas producing antibodies against surviving were obtained. IgG isotypes of two mAbs were IgG2a and IgG2b. Affinity contants were S1 1.52 × 10^-9mol/L, S2 2.31 × 10^-9 mol/L. Western blot showed that the antibodies were specific for survivin. Survivin expression in MG63 cells was proved in cell plasma by immunostaining. Conclusion The survivin mAb prepared by using recombinant survivin as antigen can be used for survivin immunoassay and in experimental treatment of osteosarcoma.