| 重组蓖麻毒素A链表达及其与天然蓖麻毒素毒性比较研究 |
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| 引用本文： | 曲英龙,钱爱东,钱军,计越,郑关雨,郭振东,赵思言,付莹莹,张毅,赵红艳,陈龙,刘林娜.重组蓖麻毒素A链表达及其与天然蓖麻毒素毒性比较研究[J].中国畜牧兽医,2015,42(4):950-957. |
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| 作者姓名： | 曲英龙 钱爱东 钱军 计越 郑关雨 郭振东 赵思言 付莹莹 张毅 赵红艳 陈龙 刘林娜 |
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| 作者单位： | 1. 吉林农业大学动物科学技术学院, 长春 130118;2. 军事医学科学院军事兽医研究所, 吉林省人兽共患病预防与控制重点实验室, 长春 130122;3. 吉林省畜牧总站, 长春 130062 |
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| 基金项目： | 国家自然基金(31101858);总后科研重大项目(AWS12J005) |
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| 摘 要： | 本试验旨在制备有高生物活性的重组蓖麻毒素A链(r-RTA)并与天然蓖麻毒素(n-RT)进行毒性比较。根据NCBI公布的RTA序列合成基因片段,并将其克隆于pET-28a载体后,利用大肠杆菌进行原核表达,镍柱亲和层析纯化上清,500mmol/L咪唑溶液洗脱获得可溶性r-RTA蛋白,通过SDS-PAGE及Western blotting对其进行鉴定并用ELISA测定其免疫原性后,对r-RTA与n-RT进行动物和细胞试验毒性比较研究。结果显示,该r-RTA在上清中表达率为31.2%;每升细菌培养物纯化后可得20mg目的蛋白,纯度≥90%,大小为32ku。其免疫原性约为n-RT的1.27倍;通过获取的n-RT细胞半抑制浓度(IC50为0.01μg/mL)及动物半数致死量(LD50为(3.27±0.44)μg/kg),以相同浓度进行细胞感染和动物攻毒试验,结果显示,同等剂量下,在细胞试验中,n-RT毒力是r-RTA的2 700倍;在动物试验中,n-RT组对动物致死率为40%,r-RTA组动物无死亡。结果表明,单独RTA,在没有RTB协助下,具有一定的毒性作用,但毒力将显著降低。该结果将为开发基于RTA的蓖麻毒素疫苗提供重要数据和理论支撑。
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| 关 键 词： | 蓖麻毒素A链(RTA) 原核表达 天然蓖麻毒素(n-RT) 毒性比较 |
| 收稿时间： | 2014-11-05 |
Study on Expression of Recombinant Ricin Toxin A Chain (RTA) and its Toxicity Compared with the Natural Ricin Toxin (RT) |
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| QU Ying-long,QIAN Ai-dong,QIAN Jun,JI Yue,ZHENG Guan-yu,GUO Zhen-dong,ZHAO Si-yan,FU Ying-ying,ZHANG Yi,ZHAO Hong-yan,CHEN Long,LIU Lin-na.Study on Expression of Recombinant Ricin Toxin A Chain (RTA) and its Toxicity Compared with the Natural Ricin Toxin (RT)[J].China Animal Husbandry & Veterinary Medicine,2015,42(4):950-957. |
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| Authors: | QU Ying-long QIAN Ai-dong QIAN Jun JI Yue ZHENG Guan-yu GUO Zhen-dong ZHAO Si-yan FU Ying-ying ZHANG Yi ZHAO Hong-yan CHEN Long LIU Lin-na |
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| Institution: | 1. College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China;2. Key Laboratory of Jilin Province Animal Disease Prevention and Control, Military Institute of Veterinary Science, Academy of Military Medical Science, Changchun 130122, China;3. Animal Husbandry Station of Jilin Province, Changchun 130062, China |
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| Abstract: | The study was aimed to produce recombinant ricin A chain (r-RTA) with high biological activity and compare its toxicity with native ricin (n-RT). We synthesized the published RTA gene sequence on NCBI and cloned it into the pET-28a vector, and then prokaryoticly expressed by the E. coli. After purification with Ni2+-NTA resin column, the r-RTA was eluted by 500 mmol/L of imidazole solutions. The purified protein was identified by SDS-PAGE and Western blotting, and the immunogenicity was determined by ELISA. Animal experiments and cell toxicity analysis were conducted to compare the toxicity between the r-RTA and n-RT. The rate of recombinant expression was 31.2%. About 20 mg fusion proteins were obtained from 1 000 mL cultures with the protein purity of ≥90%. The results of ELISA showed that the immunogenicity of r-RTA were about 1.27 times of that of n-RT. The half inhibitory concentration (IC50) of n-RT to RAW264.7 cells was 0.01 μg/mL and the median lethal dose (LD50) to mouse was (3.27±0.44) μg/kg. We used the same dose of toxin to challenge the Raw264.7 cells or mouse, and found that the virulence of n-RT was 2 700 times of that of the r-RTA. The mortality rate of n-RT was 40%, while r-RTA was all survive. Our results suggested that single RTA, without the assistance of RTB, had a significantly reduced toxicity, which provided vital data and theoretical supports for the development of RTA-based vaccine. |
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| Keywords: | ricin toxin A chain (RTA) prokaryotic expression natural ricin toxins (n-RT) comparison of toxicity |
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