论文部分内容阅读
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 preference of Pichia pastoris and without changing the primary structure of the toxin protein, the insect neurotoxin BjαIT gene was designed and synthesized and cloned into E. coli Escherichia coli fusion expression vector pPET30-a (+) and Pichia pastoris secretion Expression vector pPIC9K. Neurotoxin was expressed in E.coli under the induction of IPTG. The expressed product was purified by nickel affinity chromatography. The purified product was used to prepare antisera and activity test. The yeast transformants secreting recombinant BjαIT at a high level were screened by dot blot hybridization. Under shake flask conditions, the maximum level of toxin expression reached about 20 mg / L. E. coli BjαIT expression product was not active against Locusta migratoria manilensis and Blattela germanica, but the yeast expression product showed insecticidal activity after injection of Locusta migratoriae and Blattella germanica.