创伤弧菌溶细胞素中类蓖麻毒素功能模序的细胞毒活性研究  

Study on the Cell Toxicity of the Lectin-like Domain of RvvhA

作　　者：许玲[1] 谢旦立[1] 张刘丽[1] 楼永良[1] 

机构地区：[1]温州医学院检验医学院生命科学学院,温州325035

出　　处：《中国细胞生物学学报》2013年第1期53-58,共6页Chinese Journal of Cell Biology

基　　金：浙江省自然科学基金(批准号:Y2090468)资助的课题~~

摘　　要：创伤弧菌溶细胞素(Vibrio vulnificus cytolysin,VVC)氨基酸序列的313-445位(Gi No.M34670)与蓖麻毒素B链(Ricinchain B,RTB)的一段高度同源,为类蓖麻毒素。基因克隆表达创伤弧菌溶细胞素类蓖麻毒素(rRicin B),Ni2+亲和层析法及透析复性法进行纯化、复性。用纯化的rRi-cin B免疫日本大耳兔获得多克隆抗体,兔抗rRicin B多克隆抗体经盐析法、分子筛纯化后,得到较纯的IgG分子。免疫印迹结果显示,抗rRicin B多克隆抗体与纯化抗原呈现特异性反应条带。流式细胞术结果显示,rRicin B诱导细胞凋亡呈剂量依赖关系,多抗能够阻断其细胞毒性。至此,该工作成功制备了兔抗rRicin B的多克隆抗体,并证明了创伤弧菌溶细胞素结构基因的类蓖麻毒素功能域具有细胞毒活性,能诱导Hela细胞凋亡,且其细胞毒活性能被其多抗成功阻断。VVC amino acid sequence 313-445 （Gi No. M34670） is highly homologous to the B chain of ricin. We obtained the purified rRicin B proteins by gene cloning, purification, renaturation and purification. Rabbit polyclonal antibody against rRicin B was prepared by immunning rabbit and was purified by persulfate and Sepha- dex G25 followed by analysis of Western blot hybridization and antibody titer. Observation of the effect of rRicin B on cell apoptosis and rabbit polyclonal antibody （Endnotes） protection by flow cytometry, we found that rabbit polyclonal antibody against rRicin B was purified successfully by methods of Persulfate and Sephadex G25. Poly- clonal antibody showed high immuno-reactivity. The results of flow cytometry observation indicated that rRicin B can inhibit the growth of Hela cells and the effect was depended on doses of rRicin B. Polyclonal antibody could block up the damage of Hela cells acted by rRicin B. All the results showed that rabbit polyclonal antibody against rRicin B was prepared successfully with cell toxicity, but the cell toxicity of rRicin B could be blocked by its poly- clonal antibodies.

关 键 词：创伤弧菌 溶细胞素 类蓖麻毒素模序 多克隆抗体 凋亡 

分 类 号：R363[医药卫生—病理学]