中国寄生虫学与寄生虫病杂志 ›› 2003, Vol. 21 ›› Issue (4): 8-223.

• 论著 • 上一篇    下一篇

日本血吸虫重组信号蛋白14-3-3的纯化及抗体制备

李锋,胡敏,沈继龙   

  1. 武汉大学人民医院检验科,安徽医科大学病原生物学教研室,安徽医科大学硕士研究生 武汉 430060,合肥 230032
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2003-08-30 发布日期:2003-08-30

Purification of Recombinant Schistosoma japonicum Signaling Protein 14-3-3 and Antibody Preparation

LI Feng,HU Min,SHEN Ji-long   

  1. Clinical Laboratory,Renmin Hospital of Wuhan University,Wuhan 430060 Department of Microbiology and Parasitology,Anhui Medical University,Hefei 230032
  • Received:1900-01-01 Revised:1900-01-01 Online:2003-08-30 Published:2003-08-30

摘要:   目的制备日本血吸虫(中国大陆株)信号蛋白Sj14-3-3的多克隆抗体与单克隆抗体。方法将合Sj14-3-3重组蛋白的凝胶条带冻干磨粉,免疫家兔,制备抗Sj14-3-3多克隆抗血请;用电洗脱纯化的Sj14-3-3免疫BALB/c小鼠,用杂交瘤技术制备抗Sj14-3-3单克隆抗体。测定所得抗体效价及特异性鉴定。结果获得大量纯化的表达产物;制备的多克隆抗血清双扩效价达1:8~1:64。获得1株能稳定分泌抗Sj14-3-3单抗的杂交瘤细胞株4D9,单抗亚类为IgG1。此株单抗能与重组Sj14-3-3蛋白发生特异性反应。结论获得了高度敏感、特异的抗Sj14-3-3多克隆抗血清及稳定分泌抗Sj14-3-3单抗的杂交瘤细胞。

关键词: 日本血吸虫, Sj14-3-3蛋白, 单克隆抗体

Abstract:  Objective To prepare and characterize the polyclonal and monoclonal antibodies
McAbs against Schis- tosoma japonicum(Sj) signaling protein 14-3-3. Methods Recombinant Sj14-3-3 protein was expressed and the gel slices were lyophilized. Rabbits were immunized with the ground gel powder to prepare polyclonal antibodies against Sj14-3-3. BALB/c mice were immunized with recombinant Sj14-3-3 purified by electroelution. The anti-rSj14-3-3 monoclonal anti- body was obtained by using hybridoma technique. Characterization of the antibodies was performed by enzyme linked im- munosorbent assay (ELISA) and Western blotting analysis. Results Purified recombinant Sj14-3-3 protein was ob- tained. The titers of immune sera from the animals were 1: 8-1: 64. The monoclonal antibody(McAb) against 14-3-3 antigen was obtained and its subclass was found to be IgG1. The McAb reacted strongly and specifically with the expressed Sj14-3-3 protein. Conclusion The anti-14-3-3 sera were obtained and the hybridoma cell lines which secreted stable monoclonal antibodies against Sj14-3-3 were established, which provides a new approach to study the role of Sj14-3-3 in the signal transduction of S. japonicum.

Key words: Schistosoma japonicum, Sj14-3-3 protein, monoclonal antibody