小鼠白介素-10真核表达载体的构建及表达
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摘要
目的构建小鼠白介素-10(murineinterleukin-10,mIL-10)的真核表达载体,并研究其在293T细胞中的表达。方法 RT-PCR扩增小鼠脾脏IL-10基因后,克隆到真核表达载体pcDNA3.0上,酶切和测序鉴定重组质粒的大小、序列。采用脂质体转染法将重组质粒pcDNA3.0-mIL-10瞬时转染293T细胞,用Westernblot法检测IL-10表达。结果重组质粒pcDNA3.0-mIL-10构建成功,而且转染了293T细胞中提取的蛋白可检测到活性蛋白。结论经酶切和测序鉴定重组质粒pcDNA3.0-mIL-10构建成功,并在293T细胞中成功表达活性蛋白,为进一步研究其生物学功能奠定了基础。
Objective To construct an eukaryotic expression plasmid contained murine IL-10 gene and investigate the expression of the recombinant plasmid in 293T cells. Methods The gene of murine IL-10 was obtained by RT-PCR, and then was integrated into a eukaryotic expression plasmid pcDNA3.0, and finally the recombinant plasmid was confirmed by enzyme digestion analysis and DNA sequencing. The plasmid was transfected into 293T cells by liposome transfecting technique and was identified by Western blot. Results The recombinant plasmid pcDNA3.0-mIL-10 was constructed successfully. Furthermore, the protein was extracted from 293T cells, transfected with the plasmid showed reactivity with antibody against IL-10. Conclusion The recombinant pcDNA3.0-mIL-10 was constructed successfully and was highly expressed in 293T cells, which will lay a foundation for the further study of the biologic activity of IL-10 in vivo.
Objective To construct an eukaryotic expression plasmid contained murine IL-10 gene and investigate the expression of the recombinant plasmid in 293T cells. Methods The gene of murine IL-10 was obtained by RT-PCR, and then was integrated into a eukaryotic expression plasmid pcDNA3.0, and finally the recombinant plasmid was confirmed by enzyme digestion analysis and DNA sequencing. The plasmid was transfected into 293T cells by liposome transfecting technique and was identified by Western blot. Results The recombinant plasmid pcDNA3.0-mIL-10 was constructed successfully. Furthermore, the protein was extracted from 293T cells, transfected with the plasmid showed reactivity with antibody against IL-10. Conclusion The recombinant pcDNA3.0-mIL-10 was constructed successfully and was highly expressed in 293T cells, which will lay a foundation for the further study of the biologic activity of IL-10 in vivo.
引文
1Sabat R,Grütz G,Warszawska K,et al.Biology of interleukin-10.Cytokine Growth Factor Rev,2010,21:331-344.
2Maynard CL,Weaver CT.Diversity in the contribution of inter-leukin-10to T-cell-mediated immune regulation.Immunol Rev,2008,226:219-233.
3Ryan JJ,Kashyap M,Bailey D,et al.Mast cell bomeostasis:a funda-mental aspect of allergic disease.Crit Rev Immunol,2007,27:15-32.
4李茂新,寸向农,杨开明.免疫抑制因子IL-10在肝脏疾病中的研究进展.实用医学杂志,2009,25:1528-1529.
5Ye Z,Hang H,Ho S,et al.IL-10has a distint immunoregulatory ef-fect on naive and active T cell subsets.J Interferon Cytokine Res,2007,27:1031-1038.
6Canivet C,B?hler T,Galvani S,et al.In vitro mitogen-stimulatedT-cell from hepatitis C virus-positive liver transplantation candi-dates,increases T-cell activation markers and T-cell proliferation.Transpl Immunol,2008,19:112-119.
7郭锦锦,孙万邦.IL-10受体及其信号转导研究进展.临床医学工程,2012,1(19):135-137.
8顾秋萍,白爱平.白介素-10与炎症性肠病.世界华人消化杂志,2011,19:57-61.
9魏思忱,郑国启,田树英,等.IL-10在溃疡性结肠炎患者血清中的表达及意义.河北医药,2010,32(5):49-50.
10吴蔚,王胜军,李遇梅,等.皮肌炎患者外周血CD4+T细胞中TGF-β1、IL-10mRNA的表达及临床意义.陕西医学杂志,2009,38:806-808.
11罗莉,赵春梅,魏丽,等.CD4+CD25+T细胞、IL-10在类风湿关节炎中的表达及意义.新疆医科大学学报,2007,30:1263-1265.
12周建光,杨梅,华川.IL-10与自身免疫性疾病关系的研究进展.医学综述,2012,9:2743-2745.
2Maynard CL,Weaver CT.Diversity in the contribution of inter-leukin-10to T-cell-mediated immune regulation.Immunol Rev,2008,226:219-233.
3Ryan JJ,Kashyap M,Bailey D,et al.Mast cell bomeostasis:a funda-mental aspect of allergic disease.Crit Rev Immunol,2007,27:15-32.
4李茂新,寸向农,杨开明.免疫抑制因子IL-10在肝脏疾病中的研究进展.实用医学杂志,2009,25:1528-1529.
5Ye Z,Hang H,Ho S,et al.IL-10has a distint immunoregulatory ef-fect on naive and active T cell subsets.J Interferon Cytokine Res,2007,27:1031-1038.
6Canivet C,B?hler T,Galvani S,et al.In vitro mitogen-stimulatedT-cell from hepatitis C virus-positive liver transplantation candi-dates,increases T-cell activation markers and T-cell proliferation.Transpl Immunol,2008,19:112-119.
7郭锦锦,孙万邦.IL-10受体及其信号转导研究进展.临床医学工程,2012,1(19):135-137.
8顾秋萍,白爱平.白介素-10与炎症性肠病.世界华人消化杂志,2011,19:57-61.
9魏思忱,郑国启,田树英,等.IL-10在溃疡性结肠炎患者血清中的表达及意义.河北医药,2010,32(5):49-50.
10吴蔚,王胜军,李遇梅,等.皮肌炎患者外周血CD4+T细胞中TGF-β1、IL-10mRNA的表达及临床意义.陕西医学杂志,2009,38:806-808.
11罗莉,赵春梅,魏丽,等.CD4+CD25+T细胞、IL-10在类风湿关节炎中的表达及意义.新疆医科大学学报,2007,30:1263-1265.
12周建光,杨梅,华川.IL-10与自身免疫性疾病关系的研究进展.医学综述,2012,9:2743-2745.