重组芋螺毒素GeXIVAWT的表达、纯化和鉴定

Expression,Purification and Identfication of Recombinant Conotoxin GeXIVAWT

芋螺毒素GeXIVAWT是来自食虫将军芋螺(Conus generalis Lonnaeus)毒管中,具有两对二硫键的28肽。通过化学合成这种芋螺毒素产量低、成本高、难以纯化。利用简单快速的重组表达来生产富含二硫键的芋螺毒素有可能成为有效的新途径。通过对芋螺毒素GeXIVAWT的基因进行密码子优化,人工合成引物来构建表达载体pET22b(+)/pelB-GeXIVAWT。融合了信号肽的重组芋螺毒素以包涵体的形式在大肠杆菌中获得了高效表达。利用低浓度尿素洗涤和超滤管进行纯化无组氨酸标签的重组芋螺毒素pelB-GeXIVAWT,再利用稀释复性的方法将无活性的包涵体转变成具有活性的重组芋螺毒素。复性后的重组蛋白采用反相高效液相色谱进一步纯化后进行了质谱鉴定。活性实验表明重组芋螺毒素pelB-GeXIVAWT具有抑制昆虫细胞Sf-9的生长,为研究芋螺毒素作为生物杀虫剂奠定基础。

Conotoxin GeXIVAWT is a 28-amino acid peptide containing two pairs of disulfide bond isolated from the venom of worm-hunting Conus generalis Lonnaeus. Usually, the conotoxins produced by means of chemical synthesis was low yield, and it was difficult to be purified. A simple and fast strategy of producing disulfide-rich conotoxins via recombinant expression was presented. The codons of novel conotoxin GeXIVAWT gene were optimized. Two pairs of primers were generated by chemical synthesis for construction of expression vectors. Recombinant expression vector pET22b（＋）/pelB-GeXIVAWT fused with pelB signal peptide was successfully expressed in Escherichia coli. Recombinant pelB-GeXIVAWT without His-tag was purified by low concentrations of urea and ultrafiltration tube. Inactive inclusion bodies were transformed into active recombinant conotoxins by using dilution refolding method. It was further purified using HPLC and identified by mass spectrometry. Renaturing in vitro and biological activity experiment showed that the pelB-GeXIVAWT could inhibited the growth of Spodoptera frugiperda 9 （Sf-9） cell, as a foundation for the study of efficient insecticides.