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目的:建立检测炭疽毒素保护性抗原(PA)与靶细胞受体(ATR)结合能力的Binding-ELISA方法,并对两者结合特征进行分析。方法:以ELISA实验为基础,建立PA与ATR结合的体外模型,并对实验中的反应条件进行优化。采用Binding-ELISA方法,通过测定不同包被蛋白、不同二价阳离子条件下的D450以及相对饱和度等参数,分析PA与两种ATR的结合特征。结果与结论:以重组PA作为包被抗原时灵敏度更高,包被浓度为2μg/ml;二价阳离子浓度为5 mmol/L。两种受体与PA的亲和力不同,其中CMG2与PA的结合能力更强;并且两者与PA结合时离子依赖特征不同,PA与TEM8的结合能力在Mn2+存在时最强,PA与CMG2的结合能力在Ca2+存在时最强。这一结果与国外报道相符,进一步说明Binding-ELISA方法用于定性分析炭疽毒素与其受体结合能力的可行性,为评价以抑制PA与ATR结合为靶点的毒素抑制剂提供了有效的手段。
OBJECTIVE: To establish a Binding-ELISA method to detect the binding ability of anthrax toxin protective antigen (PA) to target cell receptor (ATR), and to analyze their binding characteristics. Methods: Based on the ELISA assay, an in vitro model of PA binding to ATR was established and the reaction conditions in the experiment were optimized. The Binding-ELISA method was used to analyze the binding characteristics of PA and the two ATRs by measuring parameters such as D450 and relative saturation for different coated proteins, different divalent cations. RESULTS AND CONCLUSION: The recombinant PA was more sensitive when coated with antigen. The coating concentration was 2μg / ml. The concentration of divalent cation was 5 mmol / L. The binding affinity of CMG2 to PA was stronger than that of PA. The ion-dependent characteristics of CMG2 and PAE were different. The binding ability of PA to TEM8 was the strongest in the presence of Mn2 + and the binding of PA to CMG2 Ability to be the strongest in the presence of Ca2 +. This result is in line with foreign reports, further illustrating the feasibility of Binding-ELISA method for the qualitative analysis of anthrax toxin binding capacity of its receptor, in order to evaluate the inhibition of PA and ATR binding target for the toxin inhibitor provides an effective means.