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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(P<0.01)。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。
Objective To investigate the effect of interfering with SelK mRNA and the expression of endoplasmic reticulum calcium homeostasis protein (CHERP) after its interference with selenoprotein K (SelK) gene. To investigate the effect of SelK mRNA on the expression of Ca 2+ in T lymphocytes Variety. METHODS: The RNAi fragment that specifically interfered with SelK mRNA was designed from the target sites in the coding sequence of SelK m RNA (NM_019979.2) to meet the design requirements. The shRNA interfering vector was designed to interfere with the targeting of SelK RNA. Nucleotide sequence analysis The effect of SelK mRNA was identified by real-time PCR and Westeron blot. Real-time PCR was used to detect the change of CHERP in cells after SelK interference. Results The positive recombinant vector can be digested with BamH I. The analysis of the sequence of the recombinant plasmid was identical with that of the designed fragment. The correct rate was 100.0%. Three ShRNAs showed different interference effects, and the interference efficiencies of sh222, sh102 and sh101 were 12.13%, 16.46% and 42.37% respectively. The interference effect of recombinant plasmid sh101 on SelK at the nucleic acid level was the best, compared with sh222 and sh102 , The difference was statistically significant (P <0.01). The interference efficiency of recombinant plasmid sh101 at the protein level was 45.4% by Western blot, and the inhibition rate of CHERP expression in the interfering cells was 40.0% compared with the control group. Conclusion The recombinant plasmid sh101 has a good interference efficiency on the level of nucleic acid and protein of SelK. The inhibition of SelK on the expression of CHERP in cells, or disrupt the balance of calcium in the endoplasmic reticulum and cytoplasm, Thus affecting the intracellular free calcium ion concentration, is one of the factors that lead to the activation of T lymphocytes.