Protective effects of TAT-tCNTF fusion protein on SH-SY5Y cells induced by β-amyloid peptide(25-35) / 中国药理学通报

ABSTRACT

Aim To determine TAT-Tcntf penetration ability and to investigate the effects of the fusion protein on SH-SY5Y cells against toxicity induced by β-amyloid peptide 25-35(Aβ_(25-35) ).Methods The conjugate(TAT-tCNTF)of TAT(47-57)of HIV-1 and the truncated human CNTF active fragment was genetic engineered and expressed in E.Coli.Immunofluorescence was used to identify cell permeation ability across membrane.MTT assay was used to measure the survival of SH-SY5Y cells injured by Aβ_(25-35).And Hoechst 33342/PI double staining was used to observe the morphology of cell apoptosis and necrosis.LDH was measured by spectrophotometric method.Results The expression vector of pBV220-TAT-tCNTF was constructed successfully.Western blot showed the recombinant fusion protein could bind specifically with CNTF antibody.The immunofluorescence assay clearly demonstrated that TAT-tCNTF did penatrate into the cells while little rhCNTF pass across the cells.Double staining and LDH release assay demonstrated that TAT-tCNTF could promote significantly the survival of the cells.Conclusion sTAT-tCNTF with high activities and effective transmembrane ability is obtained for the first time.The fusion protein protects SH-SY5Y cells from death after Aβ25-35 exposure.