少棘蜈蚣候选杀虫肽κ-SLPTX-Ssm2e的原核表达与纯化  

Prokaryotic Expression and Purification of κ-SLPTX-Ssm2e, a Candidate Insecticide from Centipede Scolopendra subspinipes mutilans

作　　者：姚梦 赵丹[1] 刘长军[1] 王干[1] 吴磊[1] 

机构地区：[1]国防科学技术大学理学院生物信息研究中心,中国湖南长沙410073

出　　处：《生命科学研究》2017年第6期477-481,共5页Life Science Research

基　　金：国防科学技术大学校预研项目(0100020414004;JC13-02-16)

摘　　要：κ-SLPTX-Ssm2e是一条通过生物信息学同源序列分析获得的与已知具有杀虫活性的毒素κ-SLPTX-Ssm2a高度同源的少棘蜈蚣毒素,由31个氨基酸残基组成,且含有3对二硫键。为了后续便于对其结构和功能进行研究,现采用原核表达与纯化的方法来获取较高产量的毒素多肽。文中首先利用大肠杆菌自动诱导表达系统,在大肠杆菌表达菌株BL21(DE3)中表达融合蛋白GST-SUMO-κ-SLPTX-Ssm2e,将其通过GST亲和层析纯化后,采用Ulp1激酶酶切,以释放毒素分子。随后,通过反相高效液相色谱(RP-HPLC)进一步纯化。最后,利用MALDI-TOF/TOF质谱仪鉴定目的多肽峰的相对分子质量为3 475.732,与κ-SLPTX-Ssm2e的理论相对分子质量3 475.07基本一致,说明κ-SLPTX-Ssm2e很有可能被成功表达。上述结果为进一步研究κ-SLPTX-Ssm2e的杀虫活性奠定了基础。The toxin κ-SLPTX-Ssm2e, from centipede Scolopendra subspinipes mutilans, is highly homologous with a known insecticidal toxin κ-SLPTX-Ssm2a. It is composed of 31 amino acid residues and three pairs of disulfide bonds. In order to explore its structure and functions, the method of prokaryotic expression was adopted to obtain abundant toxins. The recombinant protein GST-SUMO-κ-SLPTX-Ssm2e was expressed in E. coli BL21 （DE3） by auto-induction, purified by GST affinity chromatography and digested by the Ulp1 protease to release target toxin, which was further purified by reverse-phase high performance liquid chromatography （RP-HPLC）. Through the MALDI-TOF/TOF mass spectrum assay, the relative molecular mass of the polypeptide molecule was demonstrated to be 3 475.732, identical to the theoretical value 3 475.07. The research lays the foundation for further study of the insecticidal activity of κ-SLPTX-Ssm2e.

关 键 词：κ-SLPTX-Ssm2e 杀虫肽 少棘蜈蚣 原核表达 大肠杆菌BL21(DE3) 

分 类 号：Q74[生物学—分子生物学]