少棘蜈蚣候选杀虫肽κ-SLPTX-Ssm2b的原核表达与纯化

Prokaryotic Expression and Purification ofκ-SLPTX-Ssm2b,a Candidate of Insecticide from Centipede Scolopendra Subspinipes Mutilans

通过搜索NCBI数据库,获得了与具有昆虫毒性的少棘蜈蚣毒素κ-SLPTX-Ssm2a高度同源的毒素分子κ-SLPTX-Ssm2b.通过密码子优化并全基因合成获得了其DNA序列,利用RF-cloning技术将κ-SLPTXSsm2b插入到表达载体pet-HIS-SUMO,通过自动诱导表达技术在大肠杆菌BL21(DE3)诱导融合蛋白表达,通过镍柱纯化并用Ulp1激酶切割sumo标签并释放毒素分子,通过MALDI-TOF质谱鉴定其分子量为3 390.4658Da.研究结果表明获得了与理论分子量(3391.04Da)一致的κ-SLPTX-Ssm2b目的毒素分子,其表达产量为2.1mg/L,为进一步研究该候选杀虫肽分子的生理活性奠定了基础.

Searching NCBI database, k - SLPTX - Ssm2b was found possessing the similar structure of the toxin k - SLPTX- Ssm2a, isolated from the centipede Seolopendra subspinipes mutilans, and was speculated as a potential insecticide. The codingsequence was obtained by gene synthesis after codon optimization and was inserted to an expression vector pET- HIS - SUMO,which was then expressed with autoinduction method in the cytoplasm of E. coil strain BL21 （DE3）. The fusion protein waspurified using Ni - NTA column and digested by Ulpl protease to release the toxin. The released fragment was further analyzed bymass spectrum assay. It was demonstrated that the molecular weight of purified fragment （3390.4658 Da） was identicall to itstheoretical value （3391.04 Da）. The yield of expression is 2.1 rag/L, which lays the foundation for finding a new insecticidalpep fide.