expressionandpurificationoflipoproteinassociatedphospholipasea2akeyenzymeinvolvedinatherosclerosis
文献类型：期刊论文
| 作者 | Fujuan Zhang; Maojun Cai; Jingkang Shen; Yiping Wang
|
| 刊名 | actapharmacologicasinica
 |
| 出版日期 | 2006 |
| 卷号 | 27期号:6页码:679 |
| 关键词 | Lipoprotein-associated phospholipase A2 Atherosclerosis Coronary heart diseases Cloning Baculovirus Purification |
| ISSN号 | 1671-4083 |
| 英文摘要 | Aim: To express and purify lipoprotein-associated phospholipase A2 (Lp-PLA2), and to establish a screening model for Lp-PLA2 inhibitors using the expressed Lp-PLA2. Methods: We cloned the full-length cDNA of Lp-PLA2 from differentiated THP-1 cells, and subcloned the cDNA into the baculovirus transfer vector pFastBac1. In addition, we introduced an N-terminal Kozak sequence for high-level translation initiation and a C-terminal sequence of 6 histidine residues for purification. The fusion enzyme was expressed in Sf9 insect cells and purified by Ni2+ affinity chromatography. Recombinant Lp-PLA2 activity was measured using 3HPAF as a substrate, and we examined the enzyme activity of recombinant Lp-PLA2 pretreated with the known Lp-PLA2 inhibitor SB435495. Results: We successfully cloned the full-length Lp-PLA2 gene from differentiated THP-1 cells. The fusion enzyme was expressed in Sf9 insect cells at a high level and purified efficiently through a 2-step procedure. The recombinant Lp-PLA2 activity was measured using 3HPAF as a substrate, and proved to be enzymatically active. Lp-PLA2 inhibitor SB435495 produced a good inhibition curve for inhibition of recombinant Lp-PLA2 with an IC50 of 57±1 μmol/L. Conclusion: We expressed and purified Lp-PLA2 at a high level in insect cell-baculovirus expression system. The yield ratio was much greater than that obtained from human plasma and we established a screening model for Lp-PLA2 inhibitors using the expressed Lp-PLA2. |
| 语种 | 英语 |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/284206]  |
| 专题 | 中国科学院上海药物研究所 |
| 作者单位 | 中国科学院上海药物研究所 |
| 推荐引用方式 GB/T 7714 | Fujuan Zhang,Maojun Cai,Jingkang Shen,et al. expressionandpurificationoflipoproteinassociatedphospholipasea2akeyenzymeinvolvedinatherosclerosis[J]. actapharmacologicasinica,2006,27(6):679. |
| APA | Fujuan Zhang,Maojun Cai,Jingkang Shen,&Yiping Wang.(2006).expressionandpurificationoflipoproteinassociatedphospholipasea2akeyenzymeinvolvedinatherosclerosis.actapharmacologicasinica,27(6),679. |
| MLA | Fujuan Zhang,et al."expressionandpurificationoflipoproteinassociatedphospholipasea2akeyenzymeinvolvedinatherosclerosis".actapharmacologicasinica 27.6(2006):679. |
入库方式： OAI收割
来源：上海药物研究所
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