Eukaryotic expression and biological activity analysis of neuroprotective peptide Gly14-Humanin / 西安交通大学学报·英文版

ABSTRACT

Objective: To investigate the expression of neuroprotective peptide [Gly14]-Humanin (HNG) in eukaryotic cells by gene engineering technique and analyze its biological activity. Methods: By means of asymmetrical primer/template, double stranded cDNA of HNG with FLAG in its C-terminal was obtained, which was cloned into the plasmid pcDNA3.1(-), and the resultant recombinant vector pcDNA3.1(-)/HNG-FLAG was transfected into PC12 cells. At the same time, the recombinant vector pcDNA3.1 (-) /EGFP was transfected to control the efficiency of transfection. The expression of HNG in the cells was determined by immunocytochemistry. In order to analyze the biological activity of the expressed HNG, 25 μM Aβ25-35 peptide was added to the culture medium of the transfected cells for 24 h, then cell morphology, MTT assay and Hoechst 33 258 staining were observed. Results: The eukaryotic expression vector of pcDNA3.1(-)/HNG-FLAG was identified by enzyme digestion and sequencing. HNG was highly expressed in PC12 cells. After exposure of PC12 cells to 25 μM Aβ25-35 for 24 h, cell viability decreased to (65.8±5.3)% , and the dystrophic changes of neuritis and nuclei condensation were obvious. When cells were pre-transfected with pcDNA3.1(-)/HNG-FLAG, Aβ25-35-induced cell death and morphological changes of cells and nuclei were suppressed. In contrast, pre-transfected with empty vector did not protect cells from Aβ25-35-induced toxicity. Conclusion: The eukaryotic expression vector for FLAG-tagged HNG was successfully constructed and expressed in PC12 cells. Expressed HNG has biological activity.