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
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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.
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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[13]Zhang J,Li H,Yu JP,et al.Role of SOCS1 in tumor progression and therapeutic application[J].Int J Cancer,2012,130(9):1971-1980.
[14]Mansell A,Smith R,Doyle SL,et al.Suppressor of cytokine signaling 1 negatively regulates Toll-like receptor signaling by mediating Mal degradation[J].Nat Immunol,2006,7(2):148-155.
[15]Strebovsky J,Walker P,Lang R,et al.Suppressor of cytokine signaling 1(SOCS1)limits NFkappa B signaling by decreasing p65 stability within the cell nucleus[J].FASEB J,2011,25(3):863-874.
[16]Calabrese V,Mallette FA,Deschênes-Simard X,et al.SOCS1links cytokine signaling to p53 and senescence[J].Mol Cell,2009,36(5):754-767.
[17]You JM,Xu YX,Wu B,et al.Sensitivity difference of oral epithelial cells in alpha interferon between normal and lesion[J].BME&Clin Med,2015,19(1):66-71.(in Chinese)游锦梅,许雅鑫,吴彬,等.正常和病变的口腔黏膜上皮细胞对α干扰素的敏感性差异研究[J].生物医学工程与临床,2015,19(1):66-71.
[2]Judd LM,Menheniott TR,Ling H,et al.Inhibition of the JAK2/STAT3 pathway reduces gastric cancer growth in vitro and in vivo[J].PLo S One,2014,9(5):e95993.
[3]Kershaw NJ,Murphy JM,Lucet IS,et al.Regulation of Janus kinases by SOCS proteins[J].Biochem Soc Trans,2013,41(4):1042-1047.
[4]Yoshimura A,Suzuki M,Sakaguchi R,et al.SOCS,inflammation,and autoimmunity[J].Front Immunol,2012,3(1):20-28.
[5]Yamano S,Dai J,Moursi AM.Comparison of transfection efficiency of nonviral gene transfer reagents[J].Mol Biotechnol,2010,46(3):287-300.
[6]Mannava S,Grachtchouk V,Wheeler LJ,et al.Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells[J].Cell Cycle,2008,7(15):2392-2400.
[7]Endo TA,Masuhara M,Yokouchi M,et al.A new protein containing an SH2 domain that inhibits JAK kinases[J].Nature,1997,387(6636):921-924.
[8]Babon JJ,Sabo JK,Soetopo A,et al.The SOCS box domain of SOCS3:structure and interaction with the elongin BC-cullin5ubiquitin ligase[J].J Mol Biol,2008,381(4):928-940.
[9]Babon JJ,Sabo JK,Zhang JG,et al.The SOCS box encodes a hierarchy of affinities for Cullin5:implications for ubiquitin ligase formation and cytokine signalling suppression[J].J Mol Biol,2009,387(1):162-174.
[10]Yasukawa H,Sasaki A,Yoshimura A.Negative regulation of cytokine signaling pathways[J].Annu Rev Immunol,2000,18(1):143-164.
[11]Inagaki-Ohara K,Mayuzumi H,Kato S,et al.Enhancement of leptin receptor signaling by SOCS3 deficiency induces development of gastric tumors in mice[J].Oncogene,2014,33(1):74-84.
[12]Hashimoto M,Ayada T,Kinjyo I,et al.Silencing of SOCS1 in macrophages suppresses tumor development by enhancing antitumor inflammation[J].Cancer Sci,2009,100(4):730-736.
[13]Zhang J,Li H,Yu JP,et al.Role of SOCS1 in tumor progression and therapeutic application[J].Int J Cancer,2012,130(9):1971-1980.
[14]Mansell A,Smith R,Doyle SL,et al.Suppressor of cytokine signaling 1 negatively regulates Toll-like receptor signaling by mediating Mal degradation[J].Nat Immunol,2006,7(2):148-155.
[15]Strebovsky J,Walker P,Lang R,et al.Suppressor of cytokine signaling 1(SOCS1)limits NFkappa B signaling by decreasing p65 stability within the cell nucleus[J].FASEB J,2011,25(3):863-874.
[16]Calabrese V,Mallette FA,Deschênes-Simard X,et al.SOCS1links cytokine signaling to p53 and senescence[J].Mol Cell,2009,36(5):754-767.
[17]You JM,Xu YX,Wu B,et al.Sensitivity difference of oral epithelial cells in alpha interferon between normal and lesion[J].BME&Clin Med,2015,19(1):66-71.(in Chinese)游锦梅,许雅鑫,吴彬,等.正常和病变的口腔黏膜上皮细胞对α干扰素的敏感性差异研究[J].生物医学工程与临床,2015,19(1):66-71.