炭疽保护性抗原与炭疽毒素受体结合特征分析

Analysis on characteristics of binding of anthrax protective antigen and anthrax toxin receptors

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 for detecting the binding between anthrax protective antigen（PA） and anthrax toxin receptors （ATR）. Methods： The Binding-ELISA was established on the basis of the indirect ELISA under optimal experimental conditions. In the Binding-ELISA assay, via the different coated proteins or bivalent cations, the characteristics of binding of PA and ATR were analyzed by the parameter of D450 and relative saturation. Results and Conclusion： When PA was used as coated protein at 2μg/ml, and the concentration of cation was 5 mmol/L, the assay was more sensitive. The potency of binding between PA and CMG2 was more powerful in the presence of calcium cation and that of binding between PA and TEM8 was higher in the buffer containing manganous cation. Moreover, the ability of binding between PA and CMG2 exceeded that of PA and TEM8. Thus recombinant CMG2 or its excellular portion and the mutant of CMG2 may be the next-generation toxin inhibitor.The Binding-ELISA assay contributes to the research on binding mechanisms of ATR and PA and offers an effective way for evaluating the new opportunity for anthrax by restraining the binding of PA and ATR.