According to the cDNA sequence of anti neuroexcitation peptide of scorpion Buthus martensii Karsch, the putative mature anti neuroexcitation peptide (ANEP) encoding DNA fragment was obtained by PCR method, and then wa...According to the cDNA sequence of anti neuroexcitation peptide of scorpion Buthus martensii Karsch, the putative mature anti neuroexcitation peptide (ANEP) encoding DNA fragment was obtained by PCR method, and then was cloned into expression plasmid pET28a, fused with His tag at its 3’ end. When expressed in E. coli BL21 (DE3), the expression of recombinant ANEP was 15% of total cellular proteins meanwhile most recombinant ANEP products existed in the form of insoluble inclusion bodies. Co expression of molecular chaperones or protein disulfide isomerase could not improve its solubility. The recombinant ANEP in the cell lysate was purified to homogeneity by metal chelating affinity chromatography and superdex 30 chromatography. In bioassay with convulsive mice model induced by thiosemicarbazide, recombinant ANEP could apparently delay the convulsion seizure of model animals by 18% and showed anti neuroexcitatory activity.展开更多
文摘According to the cDNA sequence of anti neuroexcitation peptide of scorpion Buthus martensii Karsch, the putative mature anti neuroexcitation peptide (ANEP) encoding DNA fragment was obtained by PCR method, and then was cloned into expression plasmid pET28a, fused with His tag at its 3’ end. When expressed in E. coli BL21 (DE3), the expression of recombinant ANEP was 15% of total cellular proteins meanwhile most recombinant ANEP products existed in the form of insoluble inclusion bodies. Co expression of molecular chaperones or protein disulfide isomerase could not improve its solubility. The recombinant ANEP in the cell lysate was purified to homogeneity by metal chelating affinity chromatography and superdex 30 chromatography. In bioassay with convulsive mice model induced by thiosemicarbazide, recombinant ANEP could apparently delay the convulsion seizure of model animals by 18% and showed anti neuroexcitatory activity.
