|
|
|
| Eukaryotic Expression and Purification of Mouse CTLA-4 Outermembrane Segment |
| FANG Jing, ZHOU Ying, LI Zhi-hui, DA Fei, HU Yao-lei, MAO Jian-ping |
| Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850,China |
|
|
|
Abstract Objectives: To express the outermembrane segment of mouse CTLA-4 in eukaryotic cells. Study the peptide's binding to B7 of antigen present cells could rescue the T cells activation after stimulated, and therefor promote the T cells proliferation. Methods: The total RNA were purified from mouse spleen cells. By RT-PCR the whole gene of CTLA-4 was amplified. Primers designed according to the pcDNA3.1 and CTLA-4 outer membrane sequences, and employed for PCR. Amplified sequence together with the plasmid were cut and reconstructed. The right plasmid was further transferred into Hepa1-6 cells, and the expression was examined by western-blot. Results: CTLA-4 outer membrane segment expressing Hepa 1-6 cells were achieved. The expressed peptide were positive to anti-His antibody and CTLA-4 antibodies on western blot. Conclusion: The recombinant peptide were obtained and will be applied for cell and animal expreiments.
|
|
Received: 25 May 2010
Published: 19 November 2010
|
|
|
|
|
| [1] 郑宇,沈倍奋,黎燕,等. CTLA4-lg融合蛋白基因的表达及其生物学功能的初步研究. 细胞与分子免疫学杂志, 2000, 1(16):11-15. Zheng Y, Shen B F, Li Y, et al.Chinese Journal of Cellular and Molecular Immunology,2000,1(16):11-15.
[2] 覃林花,杨庆,卫立辛,等. 分泌mMIP-1α趋化因子的小鼠肝癌细胞株的建立及其体内致瘤性.第二军医大学学报, 2001, 22(5):13-16. Tan L H, Yang Q, Wei L X, et al. Academic Journal of Second Military Medical University, 2001,22(5):13-16.
[3] 任伟. MafA基因重组腺病毒的构建及其对小鼠肝癌细胞的影响. 内分泌外科杂志, 2008, 2(5):12-15. Ren W. Journal of Endocrine Surgery,2008,2(5):12-15.
[4] Ilka B, Annette G B. Illuminating the life of GPCRs.Cell Commun Signal,2009; 7:16-19.
[5] Amar B, Maria F, Anne G, et al. Transmembrane peptides as inhibitors of ErbB receptor signaling. Mol Biol Cell,2004,15(7):3464-3474.
[6] Norimasa K, Hiroyuki F, Yoshiya I. Primitive templated catalysis of a peptide ligation by self-folding RNAs. Nucleic Acids Res,2009,37(8): 2574-2583.
[7] Duvernay M T, Filipeanu C M, Wu G. The regulatory mechanisms of export trafficking of G protein-coupled receptors.Cell Signal,2005,17:1457-1465.
[8] Vsevolod V. Gurevich,Eugenia V. Gurevich. GPCR monomers and oligomers: it takes all kinds. Trends Neurosci,2008,31(2):74-81. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
