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目的在大肠埃希菌中可溶性表达微小隐孢子虫TSP6抗原分子,并以其纯化产物为抗原,利用杂交瘤技术生产抗TSP6单克隆抗体(McAb)。方法将pGEX-4T-1-TSP6重组质粒转化大肠埃希菌BL21(DE3),经0.1mmol/LIPTG诱导表达后超声破菌,获得可溶性表达产物,通过Gluthathione-Sepharose 4B磁珠纯化蛋白质,以纯化的重组TSP6为抗原免疫BALB/c小鼠,采用杂交瘤技术进行细胞融合,用ELISA和有限稀释法筛选分泌高滴度McAb的杂交瘤细胞株,测定其免疫球蛋白亚类及效价,Western blot分析其特异性。结果筛选出2株杂交瘤细胞能稳定分泌抗TSP6单克隆抗体,经鉴定均为IgG1。Western blot分析显示,制备的单抗均能与相应的重组蛋白特异性结合。结论制备的抗TSP6杂交瘤细胞株能分泌高特异性的McAb。
Objective To express soluble TSP6 antigen in Escherichia coli and to use its purified product as antigen to produce monoclonal antibody against TSP6 by hybridoma technology. Methods The recombinant plasmid pGEX-4T-1-TSP6 was transformed into Escherichia coli BL21 (DE3) and induced by 0.1 mmol / L IPTG. The recombinant plasmid was then sonicated and the soluble product was obtained. The purified protein was purified by Gluthathione-Sepharose 4B magnetic beads. Recombinant TSP6 was used to immunize BALB / c mice as antigen. The hybridomas were used for cell fusion. The hybridoma cell lines secreting high titer McAbs were screened by ELISA and limiting dilution. The immunoglobulin subclasses and titers were determined. Western blot analysis of its specificity. Results Two hybridoma cells were screened for the stable secretion of anti-TSP6 monoclonal antibody, both of which were identified as IgG1. Western blot analysis showed that the prepared McAb could specifically bind to the corresponding recombinant protein. Conclusion The prepared anti-TSP6 hybridoma cell lines can secrete high specificity McAb.