人成纤维细胞生长因子-21的拟南芥油体表达系统构建  

Establishment of an Arabidopsis thaliana oil body-based expression system of human fibroblast growth factor 21

作　　者：付宏岐[1,2,3] 李海燕[1,4] 庞实锋[5] 杨晶[1] 薛萍[1,4] 刘秀明[1] 王艳芳[1,2] 李营[6] 李洪志[7] 李校堃[1] 

机构地区：[1]吉林农业大学生物反应器与药物开发教育部工程研究中心,吉林长春130118 [2]吉林农业大学中药材学院,吉林长春130118 [3]渭南职业技术学院,陕西渭南714000 [4]吉林农业大学生命科学学院,吉林长春130118 [5]广东医学院生物教研室,广东东莞523808 [6]吉林市农业科学院,吉林吉林132101 [7]牡丹江医学院医药研究中心,黑龙江牡丹江157011

出　　处：《西北农林科技大学学报（自然科学版）》2011年第8期190-196,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家"863"高技术研究发展计划项目生物反应器重大专项(2007AA100503);吉林省科技发展计划重点项目(20070922);教育部高等学校科技创新工程重大项目(70S018)

摘　　要：【目的】利用拟南芥(Arabidopsis thaliana)油体系统表达人成纤维细胞生长因子-21(Fibroblastgrowth factor-21,FGF21),为利用植物油体表达系统规模化生产FGF21奠定基础。【方法】以磷酸甘露糖异构酶(PMI)基因作为转基因植物的安全筛选标记,将带有组氨酸标签的人源FGF21基因与大豆油体蛋白基因融合,克隆至大豆油体蛋白启动子驱动的表达载体pCAMBIA1390MDo(p1390MDo)上,构建植物双元表达载体p1390MDoFGF21。采用冻融法将植物双元表达载体p1390MDoFGF21转入农杆菌GV3101,花粉管导入法转化拟南芥,采用PCR检测、Southern blot筛选转基因拟南芥阳性植株(T0),并对FGF21蛋白在转基因拟南芥种子(T1)中的表达进行Western blot分析。【结果】成功构建了带PMI安全筛选标记的植物双元表达载体p1390MDoFGF21。PCR扩增和Southern blot分析结果表明,重组FGF21融合基因以单拷贝和多拷贝2种方式已整合到转基因拟南芥基因组中。BandScand 5.0软件和Western blot分析结果显示,FGF21融合蛋白约占转基因拟南芥种子可溶性蛋白的3.76%,并具有良好的免疫原性。【结论】FGF21融合基因已经转入拟南芥中,并在以PMI为选择标记的转基因拟南芥种子中成功高效表达。【Objective】 The research was done to investigate the expression of human fibroblast growth factor 21（FGF21） in Arabidopsis thaliana oil body system.【Method】 Phosphomannose isomerase gene（PMI） was used as a selection marker for transgenic plants;the plant expression vector p1390MDoFGF21 was obtained by cloning FGF21 gene into the soybean oleosin promoter-driven pCAMBIA1390MDo（p1390MDo） vector,which contained the sequence encoding the human FGF21 gene fused with oleosin and a histidine tag.After p1390MDoFGF21 was transferred into Agrobacterium tumefaciens strain GV3101,then hFGF21 gene was introduced into cultivated A.thaliana via Agrobacterium-mediated flower infection method.The positive transgenic A.thaliana plants were determind by PCR,Southern blot,and Western blot analysis.【Result】 The plant binary expression vector p1390MDoFGF21 that carries PMI selection marker was successfully constructed.Subsequent PCR and Southern blot analyses confirmed that FGF21 gene was integrated into the genome of A.thaliana plant.SDS-PAGE and Western blot analysis showed transgenic A.thaliana plant seeds had positive expression of FGF21 protein and good antigenicity.Electrophoresis strips were analyzed by BandScand 5.0 software and the FGF21 fusion protein accumulated in transgenic A.thaliana plant seeds was estimated to be approximately 3.76% of the total soluble protein.【Conclusion】 The FGF21 fusion protein was successfully expressed in transgenic A.thaliana plant seeds at relatively high levels using PMI as a selectable marker.

关 键 词：拟南芥 成纤维细胞生长因子-21 油体表达 磷酸甘露糖异构酶 

分 类 号：Q782[生物学—分子生物学]