A族链球菌表面蛋白FbaA单克隆抗体FbaAmAb2的免疫功能研究

Immune effect of FbaAmAb2 against the surface protein FbaA of group A Straptococcus

目的通过侵袭抑制及攻毒试验检测A族链球菌表面蛋白FbaA的单克隆抗体FbaAmAb2的免疫功能，为进一步探索A族链球菌的致病机制和感染后的治疗策略奠定基础。方法通过亚克隆、全菌ELISA方法筛选稳定分泌高效价FbaAmAb2的杂交瘤细胞，并将其接种于小鼠腹腔制备腹水。通过侵袭试验对FbaAmAb2能否阻止H因子与A族链球菌的结合进行研究。而后将获得的腹水倍比稀释为1：1、1：2、1：4、1：8、1：16行试管凝集试验，选定合适的稀释度被动免疫动物后用致死量A族链球菌（FbaA’）标准菌株（7．5×10’个）攻击，连续观察15d计算保护率。结果侵袭试验中FbaAmAb2能够竞争性抑制H因子与A族链球菌表面蛋白FbaA的结合，减少了H因子介导的A族链球菌侵入上皮细胞的数量。与全菌结合的ELISA效价为1：1600，试管凝集效价为1：8。在单抗被动免疫试验中，腹水原液、1：2稀释组的动物保护率高达66．67％，1：4稀释组保护率为50％，经SPSSl0．0统计软件分析各实验组与PBS组保护率差异有统计学意义（P〈0．05）。结论FbaAmAb2有望用于紧急预防及治疗A族链球菌感染，并为深入研究A族链球菌的致病机制、治疗策略及疫苗预防方面提供新的靶标。

Objective To detect the immune effect of FbaAmAb2 against the surface protein of group A Straptococcus （GAS）, and explore the pathogenesis and therapy of GAS infections. Methods By subclonal and bacterial ELISA, the positive hybridoma cells were screened that can produce better titers of FbaAmAb2 against GAS-surface FbaA protein, and were injected into the peritoneal cavities of BALB/c mice to produce ascites. The collected ascites were performed to dilute, as follows, original ascite, 1：2, 1：4, 1：8, and 1：16 to test tube agglutination. Based on the results, we selected appropriate dilution to passively immunize mice, and then challenged the mice with GAS, evaluating FbaAmAb2 neutralizing ability with GAS in mice by the survival rate of the immunized mice. Whether FbaAmAb2 could inhibit the binding of factor H to GAS was confirmed by the invasive inhibition assay. Results The IgG titer of bacteria solution ELISA is 1：160 and the titer of tube agglutination is 1：8. The protect rates of FbaAmAb2 on preventing mice with GAS infections are as follows： 66.67% in original ascite and 1：2 diluted groups, and 50% in 1：4 diluted group. Mice in each experimental group were evoked significantly protective immune responses compared with the PBS control by SPSS analysis. FbaAmAb2 can competitively inhibit factor H binding to the surface proteins FbaA of GAS, which decreased the entry of GAS into the cytoplasm of human epithelial cells through the binding of factor H. Conclusion FbaAmAb2 is promising to be used in emergent prevention or the clinical therapy for GAS infection and it is promising starting points for pharmacologic targeting and further development of new therapeutic agents for GAS.