摘要
目的构建细胞因子信号抑制因子1(suppressors of cytokine signaling 1,SOCS1)基因真核高表达质粒,并在口腔上皮细胞系NOK细胞中进行表达。方法提取健康人外周静脉血基因组DNA,PCR扩增SOCS1基因,与p EGFPN1载体连接,构建重组真核表达质粒p EGFP-N1-SOCS1,用Eco RⅠ和Bam HⅠ双酶切鉴定及测序后,转染NOK细胞,采用荧光显微镜及Western blot法检测转染细胞中SOCS1蛋白的表达。结果重组真核表达质粒p EGFP-N1-SOCS1经双酶切及测序鉴定证实构建正确。p EGFP-N1-SOCS1转染NOK细胞72 h后获得表达,SOCS1蛋白的表达量为(134.67±9.07)%,较转染空质粒组约升高4倍,二者差异有统计学意义(P=0.001)。结论成功构建了SOCS1基因真核高表达质粒,为进一步研究SOCS1的生物学功能奠定了基础。
Objective To construct a eukaryotic expression vector for suppressor of cytokine signaling 1(SOCS1) and express in oral epithelium NOK cells. Methods Genomic DNA of healthy human peripheral blood was extracted and used as a template for amplification of SOCS1 gene by PCR. The PCR product was inserted into vector p EGFP-N1,and the constructed recombinant plasmid p EGFP-N1-SOCS1 was identified by digestion with Eco R Ⅰ and Bam H Ⅰ and sequencing,with which NOK cells were transfected and observed for expression of SOCS1 protein by fluorescent microscopy and Western blot. Results Both restriction analysis and sequencing proved that recombinant plasmid p EGFP-N1-SOCS1 was constructed correctly. The expression level of SOCS1 protein in NOK cells 72 h after transfection with the recombinant plasmid was(134. 67 ± 9. 07)%,which was about 4 times higher than that in cells transfected with empty plasmid(P = 0. 001). Conclusion Eukaryotic vector for high expression of SOCS1 was successfully constructed,which laid a foundation of further study on the biological function of SOCS1.
引文
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