蝎神经毒素AaIT的表达及功能分析

Expression and Functional Analysis of a Recombinant Scorpion Neurototoxin AaIT

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 AalT 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 AalT, which was then transformed into E. coli Origami （DE3） strain. With IPTG induction, the fusion protein was expressed solubly, unfortu- nately, it had no biological activity. The AalT gene was cloned into the yeast expression vector pPlC6αA resulting in the recombinant vector pPIC6αA-AalT. The pPIC6αA-AalT plasmid was linearized with Sal Ⅰ and transformed to P. pastoris X-33 strain. Induced with methanol, the fusion protein AalT was highly secreted expressed. The recombinant toxin was biologically active against Spodoptera exigua and Argyrogramma agnata by injection.