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目的:筛选能有效中和炭疽毒素和抵抗炭疽毒素损伤细胞的CMG2-Fc(炭疽毒素受体II-人免疫球蛋白Fc段融合蛋白)突变体。方法:运用FoldX等计算软件分析CMG2与PA晶体学结构,设计能提高CMG2-PA亲和力的突变体分子,并与人IgG1Fc片段构成融合基因,转染CHO-S细胞并通过亲和层析获得CMG2-Fc突变体蛋白,通过亲和力检测和细胞保护实验分析各突变体中和炭疽毒素能力。结果:筛选并表达了8个CMG2-Fc突变体分子,亲和力实验显示其中E117Q突变可明显提高CMG2-Fc与PA的亲和力(KD=1.35×10-11 mol/L),细胞保护实验提示E117Q突变能有效提高CMG2-Fc中和炭疽毒素能力(CMG2-Fc(E117Q)的IC50为15 ng/μL,而wt CMG2-Fc的IC50为50ng/μL)。结论:CMG2-Fc(E117Q)突变体分子可作为拮抗炭疽毒素损伤的炭疽治疗药物分子,进行进一步研究。
OBJECTIVE: To screen for CMG2-Fc (anthrax toxin receptor II-human immunoglobulin Fc fusion protein) mutants that are effective in neutralizing anthrax toxin and against anthrax toxin-lesioned cells. METHODS: The crystal structure of CMG2 and PA was analyzed by FoldX software and the mutant molecules that could increase the affinity of CMG2-PA were designed and fused with human IgG1 Fc fragment. The fusion gene was transfected into CHO-S cells and obtained by affinity chromatography. -Fc mutant protein, the ability of each mutant to neutralize anthrax toxin was analyzed by affinity test and cytoprotection assay. Results: Eight CMG2-Fc mutants were screened and expressed. Affinity experiments showed that the E117Q mutation significantly increased the affinity of CMG2-Fc to PA (KD = 1.35 × 10-11 mol / L). Cytotoxicity test indicated that the E117Q mutation The IC50 for CMG2-Fc (E117Q) was 15 ng / μL, whereas the IC50 for wt CMG2-Fc was 50 ng / μL). CONCLUSION: CMG2-Fc (E117Q) mutant molecule can be used as a anthrax drug molecule against anthrax toxin damage, and further research is carried out.