新型神经保护肽[Gly14]-Humanin的表达，纯化和活性鉴定

Humanin（HN）是近年来在人体内发现的内源性多肽，能够保护神经细胞免于各种阿尔采默病病相关因素诱导的凋亡，HN肽链第14位氨基酸被Gly取代的突变体Gly14]-Humanin（HNG），神经保护活性比HN高近1 000倍。但由于HNG天然获取的途径有限，限制了对其功能的进一步研究。本课题组构建了HNG的表达质粒pET32a/HNG，并将其转化大肠杆菌BL21 trxB (DE3)，诱导表达后HNG以可溶性融合蛋白的形式出现并用镍离子亲和层析纯化，纯化的融合蛋白用肠激酶切割后经反相层析得到纯化的HNG多肽（23 mg每1 L细菌培养液），重组HNG多肽的分子量经电喷雾电离质谱（ESI-MS）检测为2876.5道尔顿，其N末端8个氨基酸残基的序列经Edman降解法测定为A-M-M-A-P-R-G-F，均与理论值一致。神经细胞保护实验表明，重组HNG多肽的神经保护作用与天然的HNG相近

Expression, Purification and Characterization of [Gly14]-Humanin, a Novel Neuroprotective Peptide

Humanin (HN, its analogue Gly14]-Humanin, HNG) was originally identified as an endogenous peptide that protects neuronal cells from apoptosis induced by various types of Alzheimer's disease-related insults. But the relative low content of this peptide in its natural sources limits its further characterization. An expression vector pET32a/HNG was corstructed and transformed it into E. coli BL21 trxB (DE3). HNG was expressed as a fusion protein in the soluble fraction and was purified by nickel affinity chromatography. Subsequently, the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC. A 23 mg recombinant HNG (rHNG) from 1 L bacterial culture was purified. The molecular weight of rHNG determined by ESI-MS was 2876.5 Da which was the expected size for correctly processed peptide. The N-terminal amino acid sequence of rHNG determined by Edman degradation method is identical to the theoretical sequence. Neuroprotective bioassay studies of rHNG exhibited its potential neuroprotective effect comparable to that of the natural HNG peptide.