蓖麻毒素A链蛋白的表达、活性及其对人肺腺癌细胞SPC的毒性作用  

Ricin A chain protein expression,activities and its toxicity to human lung adenocarcinoma cell SPC

作　　者：乔生军[1] 莫忠根[1] 林琳[1] 

机构地区：[1]上海市肿瘤研究所,上海200032

出　　处：《中国癌症杂志》2000年第1期27-30,共4页China Oncology

基　　金：上海市科学技术发展基金项目! (编号 :95 41190 2 8)

摘　　要：目的 :研究构建重组单链免疫毒素 ,对人体肿瘤进行导向治疗。方法 :采用PCR方法从克隆化载体中拉出蓖麻毒素A链 (ricinA)基因 ,装入经过改建的pTSA 18F表达载体 ,进行目的基因DNA序列分析后导入E .coliBL2 1(DE3)中进行表达 ,表达产物进行SDS PAGE和Westernblot检查 ,表达产物的生物活性通过RNAN 糖苷酶活性检查、无细胞体系中对蛋白质生物合成的抑制以及对肿瘤细胞的毒性作用来分析。结果 :SDS PAGE检查表明 ,转化子E .coliBL2 1(DE3) (pTSRA)中出现 1条Mr约 30× 10 3 大小、较空载体对照E .coliBL2 1(DE3) (pTS)明显浓黑的条带 ;Westernblot检查表明 ,转化子E .coliBL2 1(DE3) (pTSRA)中有杂交条带出现 ,而E .coliBL2 1(DE3) (pTS)中没有 ;表达蓖麻毒素A链蛋白能够特异地作用于大鼠肝 2 8S核糖体RNA ,使其产生约 45 0nt大小的核酸片段 ;对无细胞体系中蛋白质生物合成的IC50 为 3 7× 10 -9mol/L ,略高于天然蓖麻毒蛋白 ;具有与天然蓖麻毒蛋白相近的肿瘤细胞毒性。结论 :蓖麻毒素A链基因在转化子E .coliBL2 1(DE3) (pTSRA)中获得表达 ,表达产物具有与天然蓖麻毒蛋白相近的RNAN 糖苷酶活性 ,在无细胞体系中对蛋白质生物合成具有抑制作用 。Purpose:To study formation of recombinant single chain immunotoxin.Methods:Ricin A chain gene cloned in vector pUC19 was amplified by PCR and inserted into modified vector pTSA 18F for expression in E.coli BL21(DE3),the expressed ricin A protein was detected by SDS PAGE and western blot analysis and purified. The biological activities of expressed ricin A protein were analysed by RNA N glycosidase activity test and inhibition test to protein synthesis in cell free systems from rabbit reticulocyte lysate and toxicity test to cancer cells in vitro. The ability of expressed recin A protein to kill cancer cell SPC in vitro was measured by MTT method.Results:SDS PAGE showed that the heavier stained protein band with M r of up to 30?×10 3 presented in total proteins of transferant E.coli BL21(DE3)(pTSRA) while compared with E.coli BL21(DE3)(pTS) in which no target gene was contained;Western blot analysis indicated that the visible hybrid band was produced in E.coli BL21(DE3)(pTSRA),but not in E.coli BL21(DE3)(pTS); RNA N glycosidase activity test identified that the expressed ricin A protein excised mouse hepatic ribosome 28 S RNA to produce about 450 nt length fragments and had a similar RNA N glycosidase activity with natural ricin toxic protein; the inhibition of expressed ricin A protein to protein synthesis in non cell systems was present and the IC 50 was 3 7×10 -9 mol/L, a little lower than that of natural ricin toxic protein (IC 50 =0 86×10 -9 mol/L). The cell killing test in vitro showed that the expressed ricin A protein had a similar toxicity to cancer cells SPC with natural ricin toxic protein.Conclusions:Ricin A chain gene can be expressed in E.coli BL21(DE3)(pTSRA) and the expressed ricin A protein has a similar biological activities to natural ricin toxic protein.

关 键 词：蓖麻毒素A链 肺腺癌 SPC 基因表达 

分 类 号：R734.202[医药卫生—肿瘤]