CMG2-Fc突变体用于拮抗炭疽毒素损伤研究

Anthrax was an infectious disease caused by Bacillus anthracis, however, the "chief culprit" caused death was not bacillus anthracis, but anthrax toxins secreted by bacillus. There was currently no treatment for anthrax toxin damage in China. CMG2-Fc fusion protein was composed by the extracellular domain of anthrax toxin receptor 2 (capillary morphogenesis protein 2, CMG2) and Fc fragment of human IgG1, which could block anthrax toxin into cell by trap protective antigen (one component of anthrax toxins) outside the cell. The treatment,-"trapping toxin by its toxin receptor", was a good measure to deal with many pathogenic microorganisms, such as Shigellosis and Cholera toxin. The study had already proved that CMG2-Fc and CMG2-Fc(E117Q) mutation could protect F344 rats against anthrax toxins challenge, and the effect of E117Q mutant was better than CMG2-Fc. The purpose applying for this fund was further screening CMG2 mutation effectively trapped anthrax toxin, and analyzed those mutant protein structure, pharmacodynamic and pharmacokinetic characteristics.

炭疽是由炭疽芽孢杆菌引发的传染性疾病，炭疽致死的主要原因不在于炭疽杆菌，而是由细菌分泌的炭疽毒素所致，我国当前还没有针对该毒素的治疗方法。本文构建的CMG2-Fc蛋白是由炭疽毒素受体2胞外区（CMG2）和人IgG1的 Fc片段相融合，治疗原理在于通过外源性给予CMG2-Fc蛋白，竞争性干扰炭疽毒素和靶细胞膜上炭疽毒素受体结合，从而抑制炭疽毒素进入损伤细胞，这种"以可溶性毒素受体诱捕毒素"的方法也可以为其它病原微生物防治提供治疗思路。本研究前期工作已证明CMG2-Fc以及CMG2-Fc（E117Q）突变体可以有效抵抗炭疽毒素对F344大鼠的损伤，而且CMG2-Fc（E117Q）突变体更能有效抵抗炭疽毒素攻击（已申请专利）。申请该基金的目的在于根据炭疽毒素和其受体CMG2的结构特征，进一步筛选能有效拮抗炭疽毒素的CMG2-Fc突变体，并对其突变体蛋白结构、药效学和药代学特征及其相关性进行分析。

炭疽病是由炭疽杆菌感染引发的一种高致死率的疾病。CMG2-Fc是由CMG2受体结合域(vWA结构域)和人IgG1的Fc构建的融合蛋白，和当前治疗炭疽病的PA单克隆抗体相比，CMG2-Fc抗炭疽治疗不会因为炭疽杆菌PA抗原发生变异而引发治疗无效。为进一步提高CMG2-Fc中和炭疽毒素和抵抗炭疽杆菌感染的效率，我们根据CMG2-PA结合的晶体学结构特征，设计了17个CMG2-Fc突变体分子，通过CHO哺乳动物细胞表达和Protein A亲和层析获得了CMG2-Fc突变体蛋白，亲和力实验结果显示CMG2(E117Q)-Fc和CMG2(Y158Q)-Fc具有更高的亲和力，体内、外毒素中和实验显示CMG2(E117Q)-Fc具有更好的中和炭疽毒素、保护RAW264.7细胞和Fisher 344大鼠免受炭疽毒素攻击的能力，药代动力学实验结果显示CMG2(E117Q)-Fc具有更长的消除半衰期和吸收水平，组织分布数据显示CMG2-Fc主要分布于肾脏和肺脏，而其E117Q突变体主要分布于膀胱，两者都不易透过血脑屏障。上述研究结果为CMG2(E117Q)-Fc作为炭疽治疗药物的进一步开发积累基础资料。

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