昆虫神经毒素BjαIT的表达及杀虫活性

Expression and insecticidal activity of insect-specific neurotoxin BjαIT

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。

According to the codon bias of Pichia pastoris, the insect-specific neurotoxin gene BjaIT was synthesized based on its amino acid sequence and was cloned to vectors of PET-30a （ ＋ ） and pPIC9K respectively. The fusion protein of BjaIT 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.