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人TREM-1基因的克隆及其在大肠杆菌中的表达

Clone of Human TREM-1 cDNA and Its Expression in E.coli
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摘要 目的系统地研究TREM-1的生物学功能,克隆TREM-1的cDNA,构建原核表达载体后,使其在大肠杆菌中得到表达。方法用RT-PCR从临床患者外周血白细胞中扩增TREM-1 cDNA,将其克隆到克隆载体pGEM-3Z上。经酶切及PCR扩增鉴定、测序后,再克隆到原核表达载体pT7-PL上,转化大肠杆菌BL21(DE3 plys),以IPTG诱导大肠杆菌表达带有氨酸和人TREM-1蛋白组成的融合蛋白,经SDS-聚丙烯酰胺凝胶(PAGE)电泳初步鉴定,并对融合蛋白进行纯化。结果酶切电泳和DNA测序结果表明,成功地克隆了人TREM-1 eDNA;SDS-PAGE电泳结果显示相应分子质量37ku处有含人TREM-1融合蛋白的初步表达,证明了TREM-1原核表达的可行性。 Objective To clone TREM-1 cDNA, and to investigate the feasibility of prokaryotic expression of TREM-1, peripheral blood of trauma patients and healthy volunteers were collected. Methods TREM-1 cDNA was amplified by RT-PCR, cloned into vector pGZM-3Z, then amplified by PCR, authenticated by digestive enzymes, sequenced, constructed into expression vector pT7-PL, and expressed in E. coll. E. coli was induced by IPTG to express fusion protein composed of human TREM-1 protein and six lysines. The product was identified by using SDS-PAGE and purified. Results Authentication, PCR and DNA sequencing showed that the recombinant plasmid of human TREM-1 was successfully constructed. SDS-PAGE showed that human TREM-1 chimeric protein was successfully expressed in E. coli. Conclusion Our study showed that it was feasible to express TREM-1 in E. coli. Cloning of TREM-1 cDNA and its expression in E. coli facilitated determination of its biological function and further investigation of its roles in inflammation.
作者 尹燕华 李占飞 王玉 孙军 宗义强 屈伸
机构地区 华中科技大学同济医学院生物化学与分子生物学系 华中科技大学同济医学院附属同济医院创伤外科
出处 《医学分子生物学杂志》 CAS CSCD 2005年第2期98-101,共4页 Journal of Medical Molecular Biology
关键词 原核表达载体 克隆 融合蛋白 大肠杆菌表达 DNA 基因 外周血白细胞 酶切 IPTG 电泳 triggering receptors expressed on myeloid cells-1 clone expression inflammation
分类号 R392 [医药卫生—免疫学] Q78 [生物学—分子生物学]
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参考文献12

  • 1Danielson D, West MA.Recent developments in clinical management of surgical sepsis.Curr Opin Crit Care, 2001, 7(5):367-70.
  • 2Bouchon A, Dietrich J, Colonna A.Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes.J Immunol, 2000, 164(10):4 991-5.
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  • 7Allcock RJ, Barrow AD, Forbes S, et al.The human TREM gene cluster at 6p21.1 encodes both activating and inhibitory single IgV domain receptors and includes NKp44.Eur J Immunol, 2003, 33(2):567-77.
  • 8Bleharski JR, Kiessler V, Buonsanti C, et al.A role for triggering receptor expressed on myeloid cells-1 in host defense during the early-induced and adaptive phases of the immune response.J Immunol, 2003, 170(7):3 812-8.
  • 9Gingras MC, Lapillonne H, Margolin JF.TREM-1, MDL-1, and DAP12 expression is associated with a mature stage of myeloid development.Mol Immunol, 2002, 38(11):817-24.
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二级参考文献17

  • 1Danielson D , West MA. Recent developments in clinical management of surgical sepsis [J]. Curr Opin Crit Care, 2001,7(5): 367-370.
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  • 3Bouchon A, Facchetti F, Weigand MA, et al . TREM - 1 amplifies inflammation and is a crucial mediator of septic shock[J]. Nature, 2001, 410(6832): 1103 -1107.
  • 4Dapino P, Dallegri F, Quoncll L, et al. Induction of neutruphil respiratory burst by tumor necrosis factor - α: priming effect of solid - phase fibronectin and interaction of CD 11 - 18 integrins[J]. Clin Exp Immunol, 1993, 94(3): 533 -538.
  • 5Gingras MC, Lapillonne H, Margolin JF. TREM - 1 , MDL- 1 ,and DAP 12 expression is associated with a mature stage of myeloid development [J]. Mol Immuanl, 2002, 38(11): 817-824.
  • 6Bleharski JR, Kiessler V, Buonsanti C, et al . A role for triggering receptor expressed on myeloid cells- 1 in host defense during the early- induced and adaptive phases of the immune response[J]. J Immunol, 2003, 170(7): 3812 -3818.
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  • 9Bouchon A, Dietrich J, Colonna A. Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes[J]. J Immunol, 2000, 164(10): 4991- 4995.
  • 10Bouchon A, Facchetti F, Weigand MA, et al. TREM-1 amplifies inflammation and is a crucial mediator of septic shock[J]. Nature,2001, 410(6832): 1103-1107.

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医学分子生物学杂志
2005年 第2期

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