硒蛋白K基因干扰对T淋巴细胞内质网钙稳定蛋白的影响

目的探讨SelK mRNA干扰效果及其硒蛋白K(SelK)基因干扰后对细胞内质网钙稳态蛋白(endoplasmicreticulum calcium homeostasis protein,CHERP)的表达,观察T淋巴细胞内Ca~(2+)浓度的变化。方法从SelK m RNA(NM_019979.2)的编码序列中符合设计要求的靶位点设计特异性干扰SelK mRNA的RNAi片段,构建shRNA干扰载体靶向干扰SelK RNA,用核酸序列分析方法分析重组片段的正确性,利用荧光定量PCR和Westeron blot鉴定SelK mRNA的干扰效果,采用Real time-PCR的方法检测SelK干扰后细胞内的CHERP的变化。结果阳性重组载体可以被BamH I酶切。酶切片段序列分析重组子质粒结果与所设计片段完全一致,正确率为100.0%。三个ShRNA表现了不同的干扰效果,sh222、sh102和sh101的干扰效率分别为12.13%、16.46%和42.37%,在核酸水平上重组质粒sh101对SelK的干扰效果最佳,与sh222和sh102相比,差异有统计学意义(P0.01)。Western blot检测重组质粒sh101在蛋白水平上的干扰效率为45.4%,干扰组细胞中CHERP的表达与对照组相比抑制率达到40.0%。结论重组质粒sh101对SelK在核酸和蛋白质水平上都有较好的干扰效率,SelK干扰对细胞中CHERP的表达有抑制的作用,或因此打乱了内质网与胞质中的钙离子平衡,从而影响到了细胞内游离的钙离子浓度,是导致T淋巴细胞激活的因素之一。

Effect of gene interference of selenium protein K on calcium homeostasis endoplasmic reticulum protein of T lymphocytes

Objective To explore the effect of gene interference of selenium protein K (SelK) on calcium homeostasis endoplasmic reticulum protein (CHERP) of T lymphocytes,and observe the changes of Ca2+ concentration of T cells.Methods RNAi fragments were designed of specific targeted to SelK mRNA according to SelK mRNA (019979.2 NM) sequence,and shRNA interference carrier targeted SelK RNA interference was built,and the sequence of reorganization fragment was analyzed by the nucleic acid sequence,using fluorescence quantitative PCR and Western blot to analyze the interference effect of SelK mRNA with the method of Real time-PCR to detect CHERP changes in the cells after SelK interference.Results The positive recombinant vector could be cut by BamH I,the fragment sequence of enzyme-cut of recombinant plasmid was completely accorded with the design fragments,and the accuracy was 100.0％.Three ShRNAs showed different interference effects,and the interference effect rates of sh222,sh102 and sh101 were 12.13％,16.46％ and 42.37％ respectively.The interference effect of recombinant plasmid sh101 was the best at the nucleic acid level compared with sh222 and sh102,(both P＜ 0.01).The detection of recombinant plasmid sh101 interference efficiency was 45.4％ at the protein level,and the inhibition rate of CHERP express was 40.0％ in the interference group cells compared with the control.Conclusion The recombinant plasmid sh101 can interference the expression of SelK at the nucleic acid and protein levels,CHERP expression can be inhibited after the interference of SelK in cells,resulting in the disequilibrium of calcium ion in the endoplasmic reticulum and cytoplasmic,thus affecting the free calcium ion concentration of the intracellular,which is one of the factors of T lymphocyte activation.