摘要
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。
Objectives To express TSP6 of Cryptosporidium parvurn in Escherichia coli BL21 and develop monoclonal antibodies （McAb） against rCpTSP6. Methods The recombinant plasmid pGEX-4T-1 TSP6 was transformed into E. coli BL21 （DE3）. Point-one mmol/L IPTG was used to induce the expression of recombinant TSP6. The rTSP6 was sol- uble in supernatant after sonication and further purified with Glutathione-Sepharose 4B. BALB/c mice were immunized with purified proteins and cell fusion was induced via the hybridoma technique. McAbs were screened by ELISA with lim- ited dilution. The subtype and specificity of McAbs were identified by kit and Western blot analysis, respectively. Re- suits Two hybridoma cell lines secreting monoclonal antibodies against rTSP6 were obtained. The subtype of both was IgG1. Western blot analysis demonstrated that both of the McAbs reacted strongly with corresponding recombinant pro- teins. Conclusion Two hybridoma cell lines secreting highly specific McAbs against rTSP6 have been established.
出处
《中国病原生物学杂志》
CSCD
北大核心
2013年第4期325-327,306,共4页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.31072125)
