Postdoctoral Mobile Station of Biology, Genetic Engineering Research Center, College of Life Sciences, Chongqing University, Chongqing, 400030, China.
The Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province, College of Biological and Food Engineering, Huaihua University, Huaihua, 418008, China.
World J Microbiol Biotechnol. 2018 Jun 27;34(7):104. doi: 10.1007/s11274-018-2484-x.
Scorpion long-chain insect selective neurotoxin AaIT has the potential to be used against agricultural insect pests. However, there is still a lack of a heterologous gene expression system that can express AaIT efficiently. Here, using X33 as the host strain and pPICZαA as the expression vector, one transformant had the highest expression of recombinant AaIT (rAaIT) was obtained, and secreted as high as 240 mg/l rAaIT in fed-batch fermentation. Secretory rAaIT was purified by Ni-nitriloacetic affinity and CM chromatography, and 8 mg of high purity rAaIT were purified from 200 ml fed-batch fermentation cultures. Injecting silkworm (Bombyx mori Linnaeus) and Galleria mellonella larvae with rAaIT resulted in obvious neurotoxin symptoms and led to death. These results demonstrate that a large amount of anti-insect active rAaIT could be prepared efficiently.
蝎型长链昆虫选择性神经毒素 AaIT 有望用于防治农业害虫。然而，目前仍缺乏能够高效表达 AaIT 的异源基因表达系统。本研究以 X33 为宿主菌株，pPICZαA 为表达载体，获得了重组 AaIT(rAaIT)表达量最高的转化子，在分批补料发酵中可分泌高达 240mg/L 的 rAaIT。通过 Ni-亚氨二乙酸亲和和 CM 层析纯化分泌型 rAaIT，从 200ml 分批补料发酵培养物中纯化出 8mg 高纯度 rAaIT。rAaIT 对家蚕和桔小实蝇幼虫进行注射后，表现出明显的神经毒素症状，并导致死亡。这些结果表明，可以高效制备大量抗虫活性的 rAaIT。
