重组蓖麻毒素与相思子毒素B链嵌合体蛋白的制备与分析  

Preparation and analysis of a recombinant chimeric protein containing B chains of ricin and abrin

作　　者：王俊虹[1,2] 杨帆[3] 魏茂提[1,2] 徐忠伟[4] 

机构地区：[1]武警后勤学院救援医学系部队卫生统计和流行病学教研室,天津300309 [2]天津市职业与环境危害防制重点实验室,天津300309 [3]武警后勤学院基础部军事心理学教研室,天津300309 [4]武警后勤学院科研部中心实验室,天津300309

出　　处：《武警后勤学院学报（医学版）》2015年第9期696-699,共4页Journal of Logistics University of PAP（Medical Sciences）

基　　金：武警后勤学院开放基金项目(WHKF201503)

摘　　要：【目的】观察蓖麻毒素(ricin toxin,RT)、相思子毒素(abrin toxin,AT)B链的嵌合体蛋白原核表达并分析其抗原性和毒性。【方法】利用柔性linker-(G4S)3连接两个毒素B链基因(RTB和ATB),优化嵌合体基因RTB-ATB后构建表达质粒p QE80LRTB/ATB,转化至E.coli M15获得表达工程菌株,并对诱导表达条件和纯化条件进行优化;通过Western印迹检测RTB-ATB的抗原性;采用人正常肺上皮细胞Beas-2B和胃黏膜细胞GES-1进行嵌合体蛋白的细胞毒性实验。【结果】RTB-ATB全长1 647bp,编码549个氨基酸残基;E.coli M15在诱导条件为30℃、1 mmol/L IPTG,3 h后获得包涵体形式表达的目的蛋白,相对分子量约为60×103Da,经Ni柱纯化后纯度达98.9%;Western blotting印迹结果表明嵌合体蛋白保持RTB和ATB原有各自的抗原性;MTS实验结果表明目的蛋白无毒性。【结论】嵌合体蛋白RTB-ATB实现高表达,具有良好的抗原性,为研制针对RT和AT的双价疫苗奠定基础。【Objective】To observe the prokaryotic expression of the chimeric protein RTB-ATB and analyze its antigenicity and toxicity.【Methods】RTB and ATB were connected via a flexible linker（G4S）3. The expression vector p QE80L-RTB/ATB was constructed after optimizing chimeric RTB-ATB, then transformed into E. coli M15 for expression. The expression and purification conditions were optimized. The antigenicity of RTB-ATB was determined by Western blotting. Beas-2B and GES-1 were used to test the toxicity of the recombinant protein.【Results】RTB-ATB was 1 647 bp long and encoded 549 amino acid residues. After 3 h, E. coli M15 was induced to produce the target protein with MW 60×10^3Da in a condition of 30℃ and 1 mmol/L IPTG. The target protein was purified by Ni column and its purity was up to 98.9%. The results of Western blot showed that RTB-ATB was recognized by the rabbit p Ab against native RT or AT, respectively. MTS showed that RTB-ATB was non-toxic.【Conclusion】RTB-ATB is characterized by high expression and good antigenicity. It could be an effective vaccine candidate that can protect against ricin and abrin simultaneously.

关 键 词：蓖麻毒素 相思子毒素 B链 嵌合体 抗原性 

分 类 号：R994.2[医药卫生—毒理学]