细胞因子信号抑制因子1基因真核高表达质粒的构建及其在NOK细胞中的表达  

Construction of eukaryotic expression vector for suppressor of cytokine signaling 1 and its expression in NOK cells

作　　者：许雅鑫[1] 吴彬 游锦梅[3] 李钰[4] Yang Yong 陈显久[3] 

机构地区：[1]山西医科大学流行病学教研室,山西太原030001 [2]太原钢铁(集团)有限公司总医院中心实验室,山西太原030003 [3]山西医科大学生物化学与分子生物学教研室,山西太原030001 [4]山西医科大学第一医院骨科,山西太原030001 [5]Department of Epidemiology Medical Board Singapore General Hospital,singapore169608

出　　处：《中国生物制品学杂志》2015年第4期348-351,共4页Chinese Journal of Biologicals

基　　金：山西省科技(工业)攻关项目基金资助(20110321076-02);山西省国际科技合作项目基金资助(2012081050-1)

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 （SOCSI） and express in oral epithelium NOK cells. Methods Genomie 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 pEGFP-N1, and the constructed recombinant plasmid pEGFP-NI-SOCS1 was identified by digestion with EcoR I and BamH I and sequencing, with which NOK cells were transfeeted and observed for expression of SOCS1 protein by fluorescent microscopy and Western blot. Results Both restriction analysis and sequencing proved that recombinant plasmid pEGFP-NI-SOCSI was constructed correctly. The expression level of SOCSI 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 Eukaryotie vector for high expression of SOCS1 was successfully constructed, which laid a foundation of further study on the biological function of SOCS1.

关 键 词：细胞因子信号抑制因子1 真核细胞 基因表达 NOK细胞 

分 类 号：Q257[生物学—细胞生物学]