| 摘要: |
| 目的:建立表皮生长因子受体（epidermal growth factor receptor,EGFR）酪氨酸激酶(receptor tyrosine kinase,RTK)抑制剂的高通量筛选模型。方法:通过基因工程技术表达EGFR-RTK，ELISA法验证其生物学活性；应用表面等离子共振原理筛选与激酶具有结合活性的化合物，ELISA法检测其生物学活性。结果:在原核表达系统中成功表达具有生物学活性的EGFR-RTK蛋白。将蛋白偶联至生物芯片，阳性化合物EI 188与EGFR酪氨酸激酶结合的Kd值为5.00×10-7 mol·L-1，IC50为12.37 μmol·L-1，与预期结果一致。应用该模型筛选31个待测化合物，发现了6个具有结合活性和酶抑制活性的EGFR-RTK抑制剂。结论:成功建立了基于表面等离子共振原理和ELISA法的高通量EGFR-RTK抑制剂的筛选模型，为发现新型酪氨酸激酶抑制剂奠定了基础。 |
| 关键词: 表皮生长因子受体 酪氨酸激酶抑制剂 模型 高通量筛选 表面等离子共振 |
| DOI：10.3724/SP.J.1008.2009.01349 |
| 投稿时间：2009-09-04修订日期：2009-11-27 |
| 基金项目:国家自然科学基金重点项目(30330640),国家自然科学基金（30170901）,国家科技部重大科技专项(2002AA2Z3130). |
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| Establishment of high throughput screening model for epidermal growth factor receptor tyrosine kinase inhibitor |
| LI Lin1Δ,LI Guo2Δ,CHEN Li-li3,LUO Hai-bin3,CHEN Jing3,SHEN Xu3,MEI Chang-lin1* |
| (1.Department of Nephrology,Changzheng Hospital,Second Military Medical University,Shanghai 200003,China;2.Department of Orthopaedics,No. 411 Hospital of PLA,Shanghai 200081;3.Shanghai Institute of Materia Medica,Chinese Academy of Sciences,Shanghai 201203) |
| Abstract: |
| Objective:To establish a high throughput screening (HTS) model for epidermal growth factor (EGF) receptor tyrosine kinase (RTK) inhibitor. Methods: The bioactive EGFR-RTK was expressed by genetic engineering technology. The binding activity and bioactivity of the compounds were examined by surface plasmon resonance and ELISA. Results: The bioactive EGFR-RTK protein was successfully expressed in the prokaryotic expression system and was immobilized on the sensor chip. The equilibrium constants (Kd) between TKI (EI 188) and RTK was 5.00×10-7 mol·L-1 ,and the IC50 was 12.37 μmol·L-1,which was consistent with expected. With this model we screened 31 compounds and found that 6 compounds had binding activity and inhibitory activity. Conclusion: A novel HTS model of EGFR-TKI has been successfully established using surface plasmon resonance biosensor and ELISA, which lays a foundation for discovery of new TKIs. |
| Key words: epidermal growth factor receptor tyrosine kinase inhibitor model high throughout screening surface plasmon resonance |
