一种提取土壤样品中麻疯树核糖体失活蛋白curcin的方法  

A method of extracting ribosome-inactivating protein curcin of Jatropha curcas L. from soil samples

作　　者：张杨雪 蒋楠[1] 吉柔风[1] 舒苹银 冉雨露 陈放[1] 徐莺[1] 

机构地区：[1]四川大学生命科学学院,生物资源与生态环境教育部重点实验室,能源植物生物燃油制备及利用国家地方联合工程中心,成都610064

出　　处：《应用与环境生物学报》2017年第6期1129-1134,共6页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(31070301);国家“十二五”重大科技支撑计划课题(2011BAD22B08)资助~~

摘　　要：建立并优化了一种土壤麻疯树核糖体失活蛋白curcin的提取方法.优化后的提取步骤和参数为:0.1 g含curcin的土壤样品加入1 m L的提取缓冲液(5 mmol/L磷酸盐,0.1 mmol/L EDTA,0.2%SDS(m/V),20 mmol/Lβ-巯基乙醇,p H7.2),在18℃以上的室温条件下振荡混匀1 h,离心取上清液.重复该过程一次,合并上清液.上清液于4℃放置过夜,离心取上清液,即为样品粗提液.该样品粗提液即可用单克隆抗体间接酶联免疫吸附法(ELISA)进一步检测其中的curcin含量.该方法对curcin含量高于8μg/g土壤可进行定性检测,对于curcin含量分别为100、500、1 000μg/g的土壤样品的回收率分别达到49.3%±2.8%、64.2%±3.62%和78.33%±4.24%.该方法的变异系数保持在5%左右,对来自四川3个样地的土壤有较好的适用性.本研究建立优化的提取方法可为评估curcin蛋白在农业领域应用的生态风险提供技术支持.A method was established to extract curcin, one of ribosome-inactivating proteins （RIPs） from Jatropha curcas L., in soil samples. The optimized steps and parameters were as follows： 1 mL of extraction buffer （5 mmol/L phosphate, 0.1 mmol/L EDTA, 0.2% SDS [m/V], 20 mmol/L mercaptoethanol, pH 7.2） was mixed with 0.1 g of a soil sample by shaking at room temperature （above 18 ℃） for 1 h. Then, the mixture was centrifuged to obtain the supernatant. The step was repeated once. The supernatants were combined and centrifuged again to remove most of SDS after overnight incubation at 4 ℃. Then, the solution was used as a crude sample extract to detect the contents of curcin by an enzyme-linked immunosorbent assay （ELISA） with a monoclonal antibody. The method could successfully extract curcin from soil samples when its concentration was higher than 8 μg/g soil. The recovery rates were 49.3% ± 2.8%, 64.2% ± 3.62%, and 78.33% ± 4.24% when curcin contents were 100, 500, and 1 000 μg/g, respectively. The method kept its coefficient of variation within ~5% and showed good applicability to the soil samples from Sichuan Province, in China. The establishment of this method could provide technical support to assess the ecological risk of curcin in agricultural fields.

关 键 词：土壤 核糖体失活蛋白 CURCIN 麻疯树 提取方法 

分 类 号：S794.9[农业科学—林木遗传育种]