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

doi:10.16431/j.cnki.1671-7236.2015.04.027

›› 2015, Vol. 42 ›› Issue (4): 950-957.doi: 10.16431/j.cnki.1671-7236.2015.04.027

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

曲英龙^1,2, 钱爱东¹, 钱军², 计越³, 郑关雨^1,2, 郭振东², 赵思言², 付莹莹^1,2, 张毅², 赵红艳^1,2, 陈龙¹, 刘林娜²

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

收稿日期:2014-11-05 出版日期:2015-04-20 发布日期:2015-05-05
通讯作者: 刘林娜 E-mail:liulinna7@126.com
作者简介:曲英龙(1988-),女,吉林大安人,硕士生,研究方向:临床兽医学,E-mail:234041832@qq.com
基金资助:
国家自然基金(31101858);总后科研重大项目(AWS12J005)

Study on Expression of Recombinant Ricin Toxin A Chain (RTA) and its Toxicity Compared with the Natural Ricin Toxin (RT)

QU Ying-long^1,2, QIAN Ai-dong¹, QIAN Jun², JI Yue³, ZHENG Guan-yu^1,2, GUO Zhen-dong², ZHAO Si-yan², FU Ying-ying^1,2, ZHANG Yi², ZHAO Hong-yan^1,2, CHEN Long¹, LIU Lin-na²

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

Received:2014-11-05 Online:2015-04-20 Published:2015-05-05

摘要/Abstract

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,500 mmol/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20 mg目的蛋白,纯度≥90%,大小为32 ku.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0.01 μg/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.

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

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.

Key words: ricin toxin A chain (RTA); prokaryotic expression; natural ricin toxins (n-RT); comparison of toxicity

中图分类号:

Q946.1

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]. , 2015, 42(4): 950-957.

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]. , 2015, 42(4): 950-957.

参考文献

[1] Marsden C J,Knight S,Smith D C,et al.Insertional mutagenesis of ricin A chain:A novel route to an anti-ricin vaccine[J].Vaccine,2004,22(21-22):2800-2805.
[2] Lord M J,Jolliffe N A,Marsden C J,et al.Ricin.Mechanisms of cytotoxicity[J]. Toxicol Rev,2003,22(1):53-64.
[3] Gong J,Traganos F,Darzynkiewicz Z.A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry[J]. Anal Biochem,1994,218(2):314-319.
[4] Sehgal P,Kumar O,Kameswararao M,et al.Differential toxicity profile of ricin isoforms correlates with their glycosylation levels[J].Toxicology,2011,282(1-2):56-67.
[5] Gluck A,Endo Y,Wool I G.Ribosomal RNA identity elements for ricin A-chain recognition and catalysis:Analysis with tetraloop mutants[J].J Mol Biol,1992,226(2):411-424.
[6] Stirpe F,Barbieri L,Battelli M G,et al.Ribosome-inactivating proteins from plants:Present status and future prospects[J].Biotechnology (N Y),1992,10(4):405-412.
[7] Engert A,Diehl V,Schnell R,et al.A phase-Ⅰ study of an anti-CD25 ricin A-chain immunotoxin (RFT5-SMPT-dgA) in patients with refractory Hodgkin's lymphoma[J].Blood,1997,89(2):403-410.
[8] Vitetta E S,Thorpe P E.Immunotoxins containing ricin or its A chain[J]. Semin Cell Biol,1991,2(1):47-58.
[9] 孙嗣梅,王利春,康琳,等.重组蓖麻毒素A链蛋白的可溶性表达、纯化与抗原性分析[J].中国生物工程杂志,2005,25(4):47-51.
[10] Zhan J,Zhou P.A simplified method to evaluate the acute toxicity of ricin and ricinus agglutinin[J].Toxicology,2003,186(1-2):119-123.
[11] Doan L G.Ricin:Mechanism of toxicity,clinical manifestations,and vaccine development.A review[J].J Toxicol Clin Toxicol,2004,42(2):201-208.
[12] Ganguly D,Mukhopadhyay C.Steered unfolding of ricin A and B chains[J]. J Mol Graph Model,2008,27(3):266-274.
[13] Griffiths G D,Phillips G J,Holley J.Inhalation toxicology of ricin preparations:Animal models,prophylactic and therapeutic approaches to protection[J].Inhal Toxicol,2007,19(10):873-887.
[14] Bradberry S.Ricin and abrin[J]. Medicine,2012,40(2):80-81.
[15] Mantis N J.Vaccines against the category B toxins:Staphylococcal enterotoxin B,epsilon toxin and ricin[J].Adv Drug Deliv Rev,2005,57(9):1424-1439.
[16] Wilhelmsen C L,Pitt M L.Lesions of acute inhaled lethal ricin intoxication in rhesus monkeys[J].Vet Pathol,1996,33(3):296-302.
[17] Porter A,Phillips G,Smith L,et al.Evaluation of a ricin vaccine candidate (RVEc) for human toxicity using an in vitro vascular leak assay[J].Toxicon,2011,58(1):68-75.
[18] Maddaloni M,Cooke C,Wilkinson R,et al.Immunological characteristics associated with the protective efficacy of antibodies to ricin[J].J Immunol,2004,172(10):6221-6228.
[19] Marsden C J,Smith D C,Roberts L M,et al.Ricin:Current understanding and prospects for an antiricin vaccine[J].Expert Rev Vaccines,2005,4(2):229-237.
[20] Vitetta E S,Smallshaw J E,Coleman E,et al.A pilot clinical trial of a recombinant ricin vaccine in normal humans[J].Proc Natl Acad Sci USA,2006,103(7):2268-2273.
[21] Endo Y,Tsurugi K.RNA N-glycosidase activity of ricin A-chain.Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes[J]. J Biol Chem,1987,262(17):8128-8130.
[22] Olsnes S,Kozlov J V.Ricin[J]. Toxicon,2001,39(11):1723-1728.
[23] Tonevitsky A G,Toptygin A,Shamshiev A T,et al.Comparison of properties of mistletoe lectinⅠA-chain and ricin B-chain conjugate with native toxins[J].FEBS Lett,1993,336(1):100-102.
[24] Wu Y H,Shih S F,Lin J Y.Ricin triggers apoptotic morphological changes through caspase-3 cleavage of BAT3[J].J Biol Chem,2004,279(18):19264-19275.
[25] Yamasaki C,Nishikawa K,Zeng X T,et al.Induction of cytokines by toxins that have an identical RNA N-glycosidase activity:Shiga toxin,ricin,and modeccin[J].Biochim Biophys Acta,2004,1671(1-3):44-50.
[26] Zhang X,Kuča K,Dohnal V,et al.Military potential of biological toxins[J]. Journal of Applied Biomedicine,2014,12(2):63-77.
[27] 张旭,陈春,詹金彪.重组蓖麻毒素A链的表达及其对肿瘤细胞的毒性研究[J].现代实用医学,2008,20(7):518-520.

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

Study on Expression of Recombinant Ricin Toxin A Chain (RTA) and its Toxicity Compared with the Natural Ricin Toxin (RT)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	焦琳琳, 成玉婷, 吴庆国, 吴双, 吴植, 朱善元, 钱莺娟. 鸭坦布苏病毒Capsid蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2395-2402.
[2]	李灿, 李楚楚, 曾维, 张宁, 纪春晓, 陈韬. 猪RegⅢγ蛋白的重组表达及功能研究[J]. 中国畜牧兽医, 2023, 50(6): 2414-2426.
[3]	宋越, 苏胜杰, 张帆, 白帆, 戴伶俐, 王娜, 王大伟, 杨茜雯, 赵世华, 张月梅. 溶血性曼氏杆菌截短型白细胞毒素的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2479-2486.
[4]	张芳源, 蔺雅婷, 杨大为, 陈虎, 李桂梅, 单虎. 非洲猪瘟病毒P30蛋白原核表达及间接ELISA检测方法建立[J]. 中国畜牧兽医, 2023, 50(5): 2012-2022.
[5]	王雪平, 张孝俊, 李晓月, 王国栋, 张福良, 宋玉伟, 张明亮, 马磊. 高致病性禽腺病毒血清4型Hexon蛋白的原核表达及多克隆抗体的制备[J]. 中国畜牧兽医, 2023, 50(5): 2053-2060.
[6]	胡乐玉, 胡欣瑶, 李颖, 赵盛淼, 沈凤臻, 王长法, 李亮亮, 王彤彤, 任慧英. 德州驴HO-1基因生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1499-1510.
[7]	王文婕, 茹鹏辉, 郁川, 杜付熙, 陈松彪, 尚珂, 张春杰, 程相朝. 猫细小病毒洛阳分离株VP2蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1511-1521.
[8]	刘家兴, 韩雪英, 张鑫茹, 邓嘉昕, 姚伦广, 刘阳坤. 携带猪流行性腹泻病毒抗原表位的铁蛋白纳米颗粒制备与免疫原性分析[J]. 中国畜牧兽医, 2023, 50(4): 1567-1574.
[9]	贾新月, 马静, 张艳艳, 马勋, 王正荣, 薄新文. 细粒棘球绦虫EGR-07243和EGR-07244基因生物信息学分析及原核表达[J]. 中国畜牧兽医, 2023, 50(3): 847-858.
[10]	章志涛, 李祥龙, 孔令丽, 罗雨昕, 王冬英. 大片形吸虫丝氨酸/苏氨酸蛋白磷酸酶2A的原核表达及不同时期表达水平分析[J]. 中国畜牧兽医, 2023, 50(3): 1107-1117.
[11]	甘露, 郑会珍, 诺明达来, 刘燕, 金敏, 何文文, 温丽翠, 李永畅, 巴音查汗·盖力克. 伊氏锥虫伊犁株扩繁及其PFR基因克隆表达和生物信息学分析[J]. 中国畜牧兽医, 2023, 50(2): 469-478.
[12]	吕双飞, 马勋, 王静, 孔翠莲, 寇丽君, 刘彩霞, 史唯地, 康立超, 任慧杰. 炭疽芽孢杆菌PA-LF1融合蛋白的原核表达及其反应原性研究[J]. 中国畜牧兽医, 2023, 50(2): 674-683.
[13]	张凯, 张春平, 戈胜强, 孙春喜, 程杰, 伏春雨, 王刚, 牛星, 彭军. 非洲猪瘟病毒p22蛋白表达、多克隆抗体制备及间接ELISA方法的建立[J]. 中国畜牧兽医, 2023, 50(1): 270-279.
[14]	朱丽霖, 王后坤, 陈雪阳, 刘晶, 梁雄燕, 方春, 杨玉莹. 鸡IFIT5对J亚群禽白血病病毒在DF-1细胞内复制的影响[J]. 中国畜牧兽医, 2023, 50(1): 310-316.
[15]	王宇琛, 田广原, 周雅坪, 郭婷, 赵红梅, 孙雅婕, 赵逢淼, 卞宇晨, 于佳梁, 郝永清. BPIV3 NP蛋白的原核表达及其对BPIV3灭活疫苗免疫增强作用的研究[J]. 中国畜牧兽医, 2022, 49(8): 3122-3130.