Full-length cloning and 3′-terminal portion expression of human perforin cDNA

被引:5
|
作者
Li, FQ [1 ]
Zhou, XY [1 ]
Qin, WS [1 ]
Wu, JG [1 ]
机构
[1] Jinling Hosp, Ctr Med Lab Sci, Mol Biol Lab, Nanjing 210002, Peoples R China
关键词
perform; gene cloning; gene expression; hemolysis;
D O I
10.1016/S0009-8981(01)00663-5
中图分类号
R446 [实验室诊断]; R-33 [实验医学、医学实验];
学科分类号
1001 ;
摘要
Background: Perforin (also known as pore-forming protein, PFP) is one of the main effector molecules which natural killer cells (NK) and cytotoxic T lymphocytes (CTL) utilize to kill their targets both in vivo and in vitro. We report the full length of human perforin cDNA, which was cloned from liver tissue. Results: Sequencing analysis showed that there were discrepancies of four nucleotides and three amino acids compared with previously published sequence of human PFP. The cDNA fragment was then inserted into fusion protein expressive vector pGEX-2T to construct a recombinant expressive plasmid. The C-terminal truncated 125 amino acids polypeptide (410-534aa) of human perforin (hPFP-C) was selectively expressed in a form of fusion protein. Under the induction of IPTG, GST/hPFP-C fusion protein was expressed in E. coli BL21 (DE3). The fusion protein GST/hPFP-C was purified by affinity chromatography with glutathione agarose. The recombinant hPFP-C obtained by thrombin cleavage showed a significant hemolytic activity when tested with rabbit erythrocytes. Conclusion: These results suggest that the domain responsible for lytic function lies not only in the N-terminal portion but also in the C-terminal portion of perform molecule. The recombinant hPFP-C protein will be useful as a highly purified biological factor for immunological, pathological and structural studies. (C) 2001 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:125 / 131
页数:7
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