BmKANEPⅢ植物表达载体构建及其根癌农杆菌转化条件考察.docVIP

Construction of Plant Expression Vector and Transformtion for A.tumefciens of BmK ANEPⅢ Gene HUANG Ting-ting,LAI Lin-lin,WANG Yi, ZHANG Jing-hai,SONG Yong-bo （School of Biological Science and Biopharmacy, Shenyang Pharmaceutical University，Shenyang, 110016）Abstract： CONCLUSION Constructplant expression vector harboring BmK ANEPⅢ (anti-neuroexcitation peptide from Buthus martensii Karsch) gene, obtained engineering strain BI121-DE35S-ANEPⅢ/ EHA105 for genetic transformation. METHODS Using molecular cloning technique, inserted ANEPⅢ gene (added Kozak sequence to 5 end and the sequence of ER stranded peptide to 3 end) into vector pBin438, and further cloned sequences from pBin-ANEPⅢ which included CaMV DE35S promoter, Ω enhancer and ANEPⅢ gene to pBI121-Gusint-D to get pBI121-DE35S-ANEPⅢ. The recombinant plasmid was transferred into A.tumefciens EHA105 by freezing-thaw method, tri-parental mating method and electro transformation to obtained transformant. RESULTS successfully constructed plant binary expression vector pBin-ANEPⅢ and pBI121-DE35S-ANEPⅢ, obtained engineering strain pBI121-DE35S-ANEPⅢ/ EHA105. OBJECTIVE to improve our plant expression system to increase the expression level of the target protein. Key words：BmK ANEPⅢ, vector construction, plant expression vector, A.tumefciens transfection BmK ANEPⅢ植物表达载体构建及根癌农杆菌转化条件考察 黄婷婷，来琳琳，王轶，张景海，宋永波 （沈阳药科大学 生命科学与生物制药学院， 辽宁沈阳 110016） 摘要：目的 构建东亚钳蝎(Buthus martensii Karsch抗神经兴奋肽Ⅲanti-neuroexcitation peptide，ANEPⅢ)植物表达载体。方法pBin438 为母核，利用分子克隆技术构建含有启动子CaMV DE35S、Ω’翻译增强子3’端含有SEKDEL编码区的ANEPⅢ和NOS终止子表达盒，pBI121-DE35S-ANEPⅢ。EHA105，考察关键因素对转化效率的影响。结果 ANEPⅢ植物双元表达载体pBin-ANEPⅢ和pBI121-DE35S-ANEPⅢ，并获得用于遗传转化的工程菌pBI121-DE35S-ANEPⅢ/ EHA105。结论基金项目：国家人事部留学人员科技活动项目择优资助基金（编号：LXZZ007003） 作者简介：黄婷婷，女，（1983-），硕士研究生，主要从事植物基因工程药物方面的研究 通讯作者：宋永波，女，（1972-），硕士生导师，副教授，从事生物化学及生物技术制药以及蛋白质结构与功能关系方面研究。 Email address ：yongbo7200@ Tel：024东亚钳蝎抗神经兴奋肽是神经兴奋型昆虫毒素，在几种不同机理致惊厥、致癫痫动物模型及整体、离体实验动物模型中，均具较好的抗惊厥、抗癫痫作用[1～3]。一般药理及急性毒性试验结果也表明，该肽极有可能成为用于临床的一个脑神经兴