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目的为了研究脑红蛋白(neuroglobin,Ngb)发挥神经保护功能的作用机制,拟构建Ngb中关键氨基酸His突变的原核表达载体,并对其进行诱导表达与鉴定。方法利用引物突变法和PCR技术,扩增出突变型Ngb的cDNA,并克隆到原核表达载体pBV220,经测序鉴定正确转化到大肠杆菌HB101;对突变型Ngb蛋白进行诱导表达,产物经SDS-PAGE和Western印迹进行鉴定。结果与结论成功构建了pBV220-Ngb(H64V)和pBV220-Ngb(H96A)原核表达载体,SDS-PAGE和Western印迹检测结果显示突变型Ngb能够正常表达,为进一步揭示脑红蛋白神经保护功能与作用机制奠定了重要基础。
Objective To investigate the neuroprotective mechanism of neuroglobin (Ngb), we constructed a prokaryotic expression vector for the key amino acid His mutation in Ngb and induced it expression and identification. Methods The mutant Ngb cDNA was amplified by primer mutagenesis and PCR. The cDNA was cloned into prokaryotic expression vector pBV220 and transformed into E. coli HB101 by sequencing. The mutant Ngb protein was induced by SDS-PAGE And Western blotting. RESULTS AND CONCLUSION: Prokaryotic expression vectors pBV220-Ngb (H64V) and pBV220-Ngb (H96A) were successfully constructed. The results of SDS-PAGE and Western blot showed that the mutant Ngb could express normally. To further reveal the neuroprotective function of neuroglobin Mechanism has laid an important foundation.