Construction and expression of a recombinant protein with γ-aminobutyric acid type C receptor ρ2 subunits / 新乡医学院学报

ABSTRACT

Objective To construct prokaryotic expression vector of γ-aminobutyric acid type C receptor ρ2 (GABACR ρ2) gene,induce the expression of the recombination protein GABACR ρ2,and transfect the protein into SH-SY5Y cell line,achieve the transient expression of the recombination protein GABACR ρ2 in SH-SY5Y cell line.Methods The ρ2-Tat gene was inserted into plasmid pET30 to construct the recombinant plasmid pET-ρ2-GFP-Tat.The expression of GABACR ρ2 was detected by Western blot.The histidine-tagged recombination protein ρ2 was purified through immobilized Ni2+ absorption chromatographic column and the purity of GABACR ρ2 protein was detected by sodium dodecyl sulfate polyacrylamide gel elec trophoresis.The transduction and cellular localization of the GABAC R ρ2 was observed by fluorescence microscope.The SH-SY5Y cells were divided into normal group and hypoxia low glucose group.The cells in normal group were divided into normal control group which cultured in high glucose medium and without protein transfection and normal transfection group which cultured in high glucose medium and with protein transfection;the cells in hypoxia low glucose group were divided into treatment group which cultured under hypoxia low glucose condition and treatment transfection group which cultured under hypoxia low glucose and with protein transfection;the viability of SH-SY5Y cells in each group was evaluated by cell counting Kit-8.Results The recombinant plasmid pET30-ρ2-Tat was constructed successfully.The purified recombination protein ρ2-Tat successfully crossed the cytomembrane and transfected the SH-SY5Y cells.The viability of cells in normal control group,normal transfection group,treatment group and treatment transfection group was(100.0 ± 6.9)％,(89.3 ± 3.6)％,(51.4 ± 3.6)％and (66.1 ± 8.5) ％ respectively.There was no statistic difference in the cell viability between the normal control group and normal transfection group(P ＞0.05);the cell viability in treatment group was significantly lower than that in the normal control group (P ＜ 0.01);the cell viability in treatment transfection group was significantly higher than that in the treatment group (P ＜ 0.01).Conclusion The recombination protein ρ2 can through the membrane under the effect of Tat transduction peptide and can successfully establish a SH-SY5Y cell lines which transiently express recombinant protein ρ2,which provide a new research method for the study of ρ2 subunit function.