蝎神经毒素AaIT的表达及功能分析

蝎神经毒素AaIT的表达及功能分析 Expression and Functional Analysis of a Recombinant Scorpion Neurototoxin AaIT 王二文 WANG Er-wen~1 1 first-author 徐进平 XU Jin-ping~1 1 鲁伟 LU Wei~1 1 王健 WANG Jian~1 1 曹旭 CAO Xu~2 2 孟小林 MENG Xiao-lin~1 1 武汉大学生命科学学院 武汉大学生命科学学院 College of Life Sciences,Wuhan University College of Life Sciences,Wuhan University 香港绿色生命实验室 香港绿色生命实验室 Green Life Laboratory,Hongkong Green Life Laboratory,Hongkong 在不改变氨基酸编码序列的情况下,根据大肠杆菌密码子偏爱性,设计并人工合成了适合于大肠杆菌表达的蝎神经毒素AaIT基因.将该基因插入到大肠杆菌表达载体PET32a+中,得到重组质粒PET32a-AaIT,将该质粒转入带有trxB/gor双突变的大肠杆菌Origami（DE3）,重组菌株经IPTG诱导后,获得可溶性表达产物,但该表达产物没有生物学活性.把此基因插入到含有AOX1启动子和α分泌信号肽序列的毕赤酵母表达载体pPIC6αA构建重组质粒pPIC6αA-AaIT,转化毕赤酵母（X-33）,经甲醇诱导后,获得高水平的分泌表达（20 mg/L）.表达产物喂食银纹夜蛾及甜菜夜蛾没有表现出生物活性,经注射有活性. Based upon the amino acid sequences,insect-specific neurotoxin AaIT gene was designed and synthesized according to the bias in codon choice of E.coli.The AaIT gene was cloned into expression vector PET32a+ to construct the recombinant plasmid PET32a-AaIT,which was then transformed into E.coli Origami（DE3） strain.With IPTG induction,the fusion protein was expressed solubly,unfortunately,it had no biological activity.The AaIT gene was cloned into the yeast expression vector pPIC6αA resulting in the recombinant vector pPIC6αA-AaIT.The pPIC6αA-AaIT plasmid was linearized with SalⅠ and transformed to P.pastoris X-33 strain.Induced with methanol,the fusion protein AaIT was highly secreted expressed.The recombinant toxin was biologically active against Spodoptera exigua and Argyrogramma agnata by injection. 表达 AaIT 毕赤酵母 expression AaIT Pichia pastoris S476 2005-06-01 2021-04-01 6