摘要
目的构建γ-氨基丁酸受体ρ2(GABA_CR ρ2)基因的原核表达载体,诱导重组蛋白GABA_CR ρ2表达,转染SH-SY5Y细胞,实现GABA_CR ρ2亚基重组蛋白一过性表达。方法构建重组质粒p ET-ρ2-GFP-Tat;应用免疫印迹法检测GABA_CR ρ2表达;应用Ni^(2+)亲和层析柱纯化重组蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测GABA_CR ρ2蛋白纯度;荧光显微镜下观察GABA_CR ρ2的转导作用及细胞定位。将SH-SY5Y细胞分为正常组和低氧低糖组,正常组分为正常对照组(高糖培养基培养且无蛋白转染)和正常转染组(高糖培养基培养且进行蛋白转染);低氧低糖组分为处理组(低氧低糖条件下培养)和处理转染组(低氧低糖条件下培养且进行蛋白转染);使用细胞计数试剂盒检测各组细胞活力。结果重组质粒p ET30-ρ2-Tat构建正确。纯化后的重组蛋白ρ2-Tat成功透过细胞膜转染SH-SY5Y细胞。正常对照组、正常转染组、处理组和处理转染组的细胞活力分别为(100.0±6.9)%、(89.3±3.6)%、(51.4±3.6)%和(66.1±8.5)%。正常对照组与正常转染组细胞活力比较差异无统计学意义(P>0.05);处理组细胞活力显著低于正常对照组(P<0.01),处理转染组细胞活力显著高于处理组(P<0.01)。结论重组蛋白ρ2在Tat转导肽介导作用下透过细胞膜,成功建立了一过性表达重组蛋白ρ2的SH-SY5Y细胞株,为研究ρ2亚基的功能提供了新的研究方法。
Objective To construct prokaryotic expression vector of γ-aminobutyric acid type C receptor ρ2（ GABACRρ2） gene,induce the expression of the recombination protein GABACR ρ2,and transfect the protein into SH-SY5 Y cell line,achieve the transient expression of the recombination protein GABACR ρ2 in SH-SY5 Y cell line. Methods The ρ2-Tat gene was inserted into plasmid p ET30 to construct the recombinant plasmid p ET-ρ2-GFP-Tat. The expression of GABACR ρ2 was detected by Western blot. The histidine-tagged recombination protein ρ2 was purified through immobilized Ni^2＋ absorption chromatographic column and the purity of GABACR ρ2 protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The transduction and cellular localization of the GABACR ρ2 was observed by fluorescence microscope. The SHSY5 Y 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-SY5 Y cells in each group was evaluated by cell counting Kit-8. Results The recombinant plasmid p ET30-ρ2-Tat was constructed successfully. The purified recombination protein ρ2-Tat successfully crossed the cytomembrane and transfected the SH-SY5 Y 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
出处
《新乡医学院学报》
CAS
2018年第1期1-5,共5页
Journal of Xinxiang Medical University
基金
国家自然科学基金资助项目(编号:NSFC81100912)
新乡医学院研究生创新课题资助项目(编号:YJSCX20412Z)
关键词
γ-氨基丁酸C型受体
ρ2亚基
原核表达
蛋白转染
γ-aminobutyric acid type C receptor
ρ2 subunits
prokaryotic expression
protien transfection
