基于表面等离子共振技术的药物抗淀粉样蛋白聚集作用的实验方法研究

Study on drug's anti beta amyloid aggregation based on Surface Plasmon Resonance technique

目的:探索基于表面等离子共振技术的抗淀粉样蛋白聚集药物的实验条件、方法。方法:制备Amyloidβ1-40与1-42单体和聚体,配制对照分析物CR/TS/TT浓度梯度(0μg/ml、7. 81μg/ml、15. 63μg/ml、31. 25μg/ml、62. 5μg/ml、125μg/ml、250μg/ml、500μg/ml)。采用CM5芯片,分别将蛋白的单体和聚体通过氨基偶联固定于芯片表面。将样品依次进样,进行亲合分析。结果:Aβ1-40与Aβ1-42单体在25℃温度下静置24 h可制备成实验用聚集体; SPR分析系统进样参数:流速30μl/min,结合时间300s,解离时间300s,再生1 m M Na OH 15s;实验优选阳性对照为CR(Congo Red)。结论:以上实验条件可用于SPR系统中抗β淀粉样蛋白聚集药物的初步筛选,实验方法稳定,结果可信。

Objective： To explore the experimental conditions and methods of drug’s anti beta amyloid aggregation based on Surface Plasmon Resonance technique. Methods： Both monomers and fibril β-amyloid protein fragment 1-40 and 1-42 were prepared,and the CR/TS/TT with concentration gradient of 0 μg/ml、7. 81 μg/ml、15. 63 μg/ml、31. 25 μg/ml、62. 5 μg/ml、125 μg/ml、250 μg/ml、500 μg/ml were prepared. Monomers and polymers of Aβ1-40 and Aβ1-42 were fixed on the surface of the CM5 chip by amino-coupling,then the samples were injected in order to analysis the binding ability of two forms of Aβ1-40 and Aβ1-42. Results： Both Aβ1-40 and Aβ1-42 aggregate on the condition of incubating at 25℃ for 24 h without stirring. The parameters of the SPR analysis system were about ： current speed at 30μl/min,combined time is 300 s,dissociation time is 300 s,regenerate time is 15 s,and the positive control was Congo Red. Conclusion： The above experimental conditions can be used for preliminary screening of anti-β-amyloid aggregation drugs in SPR system. The experimental method is stable,and the result is credible.