期刊文献+
任意字段
题名或关键词
题名
关键词
文摘
作者
第一作者
机构
刊名
分类号
参考文献
作者简介
基金资助
栏目信息

B7-H1慢病毒的制备及其在B16F10细胞中的表达鉴定 被引量：1

Construction of recombinant lentivirol expression vector carrying mouse B7-H1 gene and its expression and identification in B16F10 cells
下载PDF
导出
摘要 目的构建小鼠B7-H1分子慢病毒表达载体并包装成感染性病毒颗粒,获得稳定表达B7-H1的B16F10细胞。方法以小鼠B7-H1的测序质粒为模板,PCR扩增B7-H1全长,装入pLenti6/V5-D-TOPO慢病毒表达载体,经过测序证实其序列与标准序列完全一致。将B7-H1表达载体用L ipofectam ineTM2000转染293FT细胞,包装成感染性病毒颗粒,感染B16F10细胞后,加入含B lastic id in(终浓度为4.0μg/m l)的RPM I1640培养基,筛选出稳定表达B7-H1蛋白的B16F10细胞株。结果PCR扩增得到编码小鼠B7-H1的877 bp基因片段,与预期大小一致。B7-H1重组慢病毒感染B16F10细胞后,用B7-H1单抗进行免疫组化检测,荧光显微镜观察显示B7-H1分子表达于B16F10细胞。FACS测定B16F10细胞荧光阳性率为36%。结论成功构建了B7-H1慢病毒表达载体,包装出感染性病毒颗粒,重组慢病毒感染B16F10细胞后表达出有活性的B7-H1膜表面蛋白,为进一步研究B7-H1分子在器官移植、自身免疫性疾病以及肿瘤免疫逃逸机制中的作用奠定了基础。 Objective To construct lentivirol expression vector of mouse B7-H1, and identify its expression in B16F10 cells. Methods Mouse B7-H1 encoding sequence was amplified, purified, ligated with pLen- ti6/V5 lentivirol vector and verified by sequencing. The verified recombinant was transfected into 293FT cells by LipofectamineTM 2000 reagent. The supernatant of the cultured 293FT cells was collected and used to infect B16F10 cells. B16F10 cell line stably expressing B7-H1 protein was selected in the presence of Blasticidin （4.0μg/ml）. Results The mouse B7-H1 gene was successfully amplified by PCR. The recombinant lentivirol vector carrying B7-H1 gene for expression was constructed and its length was about 877 bp, very close to the expected value. By transfecting package cell line 293FT, the recombinant of pLenti6/VS-D-TOPO lentivirol vector carrying B7-H1 infected B16F10 ceils in selective medium. B16F10 transgenic cells could stably express mouse B7-H1 protein on cell membrane. Conclusion Successful construction of mouse B7-H1 lentivirol expression vector and expression of its functional fusion protein based further investigation of the role of B7-H1 in immune tolerance, autoimmune disease and tumor immune escape.
作者 张立群 段文元 许雪青 黄钢 陈雪丹 白云
机构地区 第三军医大学基础医学部医学遗传学教研室
出处 《第三军医大学学报》 CAS CSCD 北大核心 2007年第1期24-27,共4页 Journal of Third Military Medical University
基金 国家自然科学基金(30271246)~~
关键词 B7-H1 慢病毒 基因治疗 B7-H1 lentivirol gene therapy
分类号 R373 [医药卫生—病原生物学] R392-33 [医药卫生—免疫学]
  • 相关文献

参考文献12

  • 1Dong H,Chem L.B7-H1 pathway and its in the evasion of tumor immunity[J].J Mol Med,2003,81(5):281-287.
  • 2Selenko-Gebauer N,Majdic O,Szekeres A,et al.B7-H1(programmed death-1 ligand)on dendritic cells is involved in the induction and maintenance of T cell ancrgy[J].J Immunol,2003,170(7):3637-3644.
  • 3Magnus T,Schreinerb ,Korn T,et al.Microglial expression of the B7-family member B7 homolog 1 confers strong immune inhibition:implications for immune responses and autoimmunity in the CNS[J]. J Neurosci, 2005, 25 (10): 2537-2546.
  • 4Kim H K,Guan H,Zu G, et al. High-level expression of B7-H1 molecules by dendritic cells suppresses the function of activated T cells and desensitizes allergen-primed animals [ J ]. J Leuk Biol, 2006, 79 ( 4 ) :686 - 695.
  • 5Greenwald R J, Freeman G J, Sharep A H. The B7 family revisited[J]. Ann Rev Immunol, 2005, 23(4): 515 -548.
  • 6Ichikawa M,Chen L. Role of B7-H1 and B7-H4 molecules in down-regulating effector phase of T-cell immunity: novel cancer escaping mechanisms[J]. Front Biosci, 2005, 10(9): 2856-2860.
  • 7Kosmaczewska A, Magott-Procelewska M, Frydecka I, et al. CD40L,CD25, and CTLA--4 expression on CD4 + T cells in kidney graft recipients: a relationship with post-transplantation clinical course [ J].Transpt Immunol, 2006, 16( 1 ) : 32 -40.
  • 8Van-Berkel M E, Oosterwegel M A. CD28 and ICOS: similar or separate costimulators of T cells [ J ] ? Immunol Lett, 2006, 105 ( 2 ) : 115 -122.
  • 9Borst J, Hendriks J,Xiao Y. CD27 and CD70 in T cell and B cell activation[J]. Curr Opin Immunol, 2005, 17 (3) : 275 - 281.
  • 10Maciej W, Didiert. Harnessing HIV for therapy, basic research and biotechnology[ J]. Trends Biotechnol, 2005, 23 ( 1 ) : 42 -47.

二级参考文献20

  • 1Nishimura H, Okazaki T, Tanaka Y, et al. Autoimmune dilated cardiomyopathyin PD-1 receptor-deficient mice [J]. Science, 2001, 291(5502): 319 - 322.
  • 2Okazaki T, Tanaka Y, Nishio R, et al. Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-l-deficient mice[J]. Nat Med, 2003, 9(12): 1477- 1483.
  • 3Salama A D, Chitnis T, Imitola J, et al. Critical role of the programmed death-1 (PD-1) pathway in regulation of experinental autoimmune encephalomyelitis[J]. J Exp Med, 2003, 198(4): 677 - 686.
  • 4Kanai T, Totsuka T, Uraushihara K, et al. Blockade of B7-H1 suppresses the development of chronic interstinal inflammation[ J]. J Immunol, 2003,171 (8): 4156-4163.
  • 5Bennett F, Luxenberg D, Ling V, et al. Programmed death-1 engagement upon TCR activatiou has distinct effects on costimulation and cytokine-driven proliferation: attenuation of ICOS, IL-4, and IL-21, but not CD28,IL-7, and IL-15 responses[J]. J Immunol, 2003, 170(2): 711- 718.
  • 6Hutloff A, Dittrich A M, Beier K C, et al. ICOS is an inducible T-cell costimulator structurally and functionally related to CD28[J]. Nature, 1999,397(6716): 263 - 266.
  • 7Yoshinaga S K, Whoriskey J S, Khare S D, et al. T-cell co-stimulation through B7RP-1 and ICOS[J]. Nature, 1999, 402(6763): 827-832.
  • 8Ozkanak E, Gao W, Shemmeri N, et al. Importance of ICOS-B7RP-1 costimulation in acute and chronic allograft rejection[J]. Nature, 2001, 2(7): 591 - 596.
  • 9Mcadam A J, Chang T, Lumelsky A L, et al. Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation andregulates differentiation of CD4+ T cells[J]. J Immunol, 2000, 165(9):5035 - 5040.
  • 10Iwai H, Kozono Y, Hirose S, et al. Amelioration of collagen-induced arthritis by blockade of inducible costimulator-B7 homologous protein costimulation[J]. J Immunol, 2002, 169(8): 4332-4339.

共引文献1

同被引文献13

  • 1De-Paula S, Greggio S, DaCosta J C. Use of stem cells in perinatal asphyxia: from bench to bedside[J]. J Pediatr (Rio J), 2010, 86(6) : 451 -464.
  • 2Marquass B, Somerson J S, Hepp P, et al. A novel MSC+seeded triphasic construct for the repair of osteochondral defects[ J]. J Orthop Res, 2010, 28(12) : 1586 -1599.
  • 3Ng S Y, Wong C K, Tsang S Y. Differential gene expressions in atrial and ventricular myocytes: insights into the road of applying embryonic stem cell-derived cardiomyocytes for future therapies[ J]. Am J Physiol Cell Physiol, 2010, 299(6) : C1234 - C1249.
  • 4Schuler D. Genetics and cell biology of magnetosome formation in mag- netotaetie bacteria [ J ]. FEMS Mierobiol Rev, 2008, 32 (4) : 654 - 672.
  • 5Arakaki A, Nakazawa H, Nemoto M, et al. Formation of magnetite by bacteria and its application [ J ]. J R Soc Interface, 2008, 5 (26) : 977 - 999.
  • 6Wang X, Liang L. Effects of static magnetic field on magnetosome for- mation and expression of mamA, mms13, mms6 and mag, A in Magneto- spirillum magneticum AMB-1 [ J ]. Bioelectromagnetics, 2009, 30 (4) : 313 -321.
  • 7Ju S, Teng G J, Lu H, et al. In vivo MR tracking of mesenchymal stem cells in rat liver after intrasplenic transplantation[ J]. Radiology, 2007, 245 ( 1 ) : 206 - 215.
  • 8Focke A, Schwarz S, Foerschler A, et al. Labeling of human neural precursor ceils using ferromagnetic nanoparticles [ J ]. Magn Reson Med, 2008, 60(6) : 1321 -1328.
  • 9Cai J, Zhang X, Wang X, et al. In vivo MR imaging of magnetically labeled mesenchymal stem cells transplanted into rat liver through hepatic arterial injection [ J ]. Contrast Media Mol Imaging, 2008, 3 (2) : 61 - 66.
  • 10Gilad A A, Winnard P T Jr, van-Zijl P C, et al. Developing MR reporter genes : promises and pitfalls [ J ]. NMR Biomed, 2007, 20 (3) : 275 -290.

引证文献1

第三军医大学学报
2007年 第1期

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部