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)

作　　者：曲英龙 钱爱东[1] 钱军[2] 计越 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] 

机构地区：[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

出　　处：《中国畜牧兽医》2015年第4期950-957,共8页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然基金(31101858);总后科研重大项目(AWS12J005)

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。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 Ni^2＋-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.

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 

分 类 号：Q946.1[生物学—植物学]