抗重组日本血吸虫P38抗原单克隆抗体的制备与鉴定  

Development and Identification of Monoclonal Antibodies against the Recombinant P38 Antigen of Schistosoma japonicum

作　　者：吴锦雅[1] 周晓红[1] 陈晓光[1] 

机构地区：[1]南方医科大学寄生虫学教研室,广州510515

出　　处：《中国寄生虫学与寄生虫病杂志》2005年第2期110-113,共4页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：WHO/TDR研究基金资助(A990310;A00191)~~

摘　　要：目的在大肠埃希菌中可溶性表达日本血吸虫P38(SjP38)抗原分子,并以其纯化产物为抗原,制备抗SjP38单克隆抗体(McAb)。方法将pET32(a)-P38重组质粒转化大肠埃希菌BL21(DE3),在1mmol/L异丙基-β-D-硫代半乳糖苷诱导下表达,超声破菌后获得可溶性表达产物,通过镍柱一步法纯化,以纯化的重组SjP38为抗原,免疫BALB/c小鼠,采用杂交瘤技术制备McAb,用ELISA和有限稀释法筛选出高分泌滴度McAb杂交瘤细胞株,测定其免疫球蛋白亚类及其效价,蛋白质印迹法(Westernblotting)分析其特异性。结果筛选出能稳定分泌抗重组SjP38单克隆抗体的8株杂交瘤细胞株,均为IgG1;Westernblotting分析显示8株单抗能与日本血吸虫虫卵抗原中的天然P38发生特异性结合。结论制备的抗P38杂交瘤细胞株能分泌高滴度、高特异性的McAb。Objective To express P38 of Schistosoma japonicum in Escherichia coli BL21 and develop the monoclonal antibodies (McAb) against rSjP38. Methods The recombinant plasmid pET32(a)-P38 was transformed into E.coli BL21(DE3). 1 mmol/L IPTG (isopropyl-beta D-thiogalactopyranoside) was used to induce the expression of the recombinant rSjP38. The rSjP38 was soluble in supernatant after sonication and further purified by His-Ni chromatography. BALB/c mice were immunized with the purified rSjP38 and hybridomas were generated with traditional technique. McAbs were screened by ELISA with limited dilution. The subtype and specificity of McAb were identified by kit and Western blot respectively. Results Eight hybridoma cell lines secreting monoclonal antibodies to rSjP38 were obtained. The subtype of all the 8 McAbs are IgG1. Western blotting showed that the 8 McAbs reacted strongly and specifically with native antigen (P38) of Schistosoma japonicum. Conclusions Eight hybridoma cell lines secreting highly specific McAbs against P38 have been established.

关 键 词：单克隆抗体 重组日本血吸虫 制备 38抗原 BL21(DE3) 杂交瘤细胞株 BALB/c小鼠 blotting 免疫球蛋白亚类 Western 大肠埃希菌 鉴定 McAb 蛋白质印迹法 可溶性表达 mol/L 有限稀释法 ELISA 杂交瘤技术 特异性结合 抗原分子 

分 类 号：R392-33[医药卫生—免疫学] R383.24[医药卫生—基础医学]