单 位：
重庆大学生物工程学院基因工程研究中心;重庆市杀虫真菌农药工程技术研究中心;重庆市高校功能基因及调控技术重点实验室
关键词：
昆虫神经毒素;BjαIT;大肠杆菌;毕赤酵母;融合表达;东亚飞蝗;德国小蠊;杀虫活性
摘 要：
根据毕赤酵母Pichia pastoris密码子偏爱性,不改变毒素蛋白质一级结构,设计合成了昆虫神经毒素BjαIT基因,并分别克隆至大肠杆菌Escherichia coli融合表达载体pPET30-a(+)和毕赤酵母分泌表达载体pPIC9K。在IPTG的诱导下,神经毒素在大肠杆菌中融合表达,表达产物利用镍亲和层析柱纯化,纯化产物用于制备抗血清和活性测试。采用斑点杂交,筛选得到了较高水平分泌表达重组BjαIT的酵母转化子,摇瓶条件下,毒素表达量最大可达约20mg/L。大肠杆菌BjαIT表达产物对东亚飞蝗Locusta migratoria manilensis和德国小蠊Blattela germanica没有活性,但酵母表达产物经注射东亚飞蝗和德国小蠊表现出杀虫活性。
译 名：
Expression and insecticidal activity of insect-specific neurotoxin BjαIT
作 者：
LI Hong-Bo,XIA Yu-Xian(Genetic Engineering Research Center,Bioengineering College of Chongqing University/Chongqing Engineering Research Center for Fungal Insecticides/Key Laboratory of Functional Gene and Regulation Technologies under Chongqing Municipal Education Commission,Chongqing 400030,China)
关键词：
Insect-specific neurotoxin; BjαIT; Escherichia coli; Pichia pastoris; fusion expression;Locusta migratoria manilensis; Blattela germanica;insecticidal activity
摘 要：
According to the codon bias of Pichia pastoris,the insect-specific neurotoxin gene BjαIT was synthesized based on its amino acid sequence and was cloned to vectors of PET-30a (+) and pPIC9K respectively. The fusion protein of BjαIT expressed in Escherichia coli was induced with IPTG and was purified with Ni-NTA His Bind Column. The purified fusion protein was used to prepare antiserum and conduct bioactivity test. Dot blotting was used to screen the high-level expressed transformants of P. pastoris. The results showed that the highest expression of recombinant BjαIT in P. pastoris was about 20 mg/L in baffled flasks,and the BjαIT fusion protein expressed in E. coli was not toxic to locust Locusta migratoria manilensis and cockroach Blattela germanica,but that expressed in P. pastoris had insecticidal activity against locust and cockroach through injection.
