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目的制备抗GⅡ.17型诺如病毒(noroviruses,No V)单克隆抗体,并鉴定其生物学特性。方法将小鼠的骨髓瘤细胞SP2/0与经GⅡ.17 No V VLPs抗原免疫的BALB/c小鼠的脾细胞进行融合,通过替代性的中和试验筛选出阳性杂交瘤细胞,制备单克隆抗体并进行纯化,ELISA法检测单克隆抗体效价,SDS-PAGE法检测单克隆抗体纯度,BCA法检测单克隆抗体浓度,Biacore法检测单克隆抗体亲和力,亚型试剂鉴定单克隆抗体亚型,并进行各单克隆抗体与不同型NOV VLPs-唾液人类组织血型抗原(histo-blood group antigen,HBGA)的交叉中和试验,单抗与不同型NOV VLPs结合能力的Western blot鉴定。结果筛选出3株具有中和活性的细胞株,分别命名为E12、B7、H7,纯化后的3株单克隆抗体效价均在106以上,浓度分别为6.932、2.662和2.371 mg/ml,亲和力常数KD均为10-9 mol/L,单克隆抗体E12的纯度在93%以上,属于Ig G2a亚型,单克隆抗体B7和H7的纯度分别在98%以上,属于Ig G1亚型。3株单克隆抗体仅对GⅡ.17型No V具有中和活性,对其他型的No V无中和活性,且与8种抗原均发生结合反应,但E12对8种抗原的结合能力较B7、H7差。结论已成功制备出抗GⅡ.17 No V的单克隆抗体,为进一步研究该病毒的生物学特性及疫苗的开发奠定了基础。
Objective To prepare anti-GⅡ.17 noroviruses (No V) monoclonal antibody and identify its biological characteristics. Methods The mouse myeloma cells SP2 / 0 were fused with the spleen cells of BALB / c mice immunized with the GII.17 No V VLPs antigen. The positive hybridoma cells were screened by alternative neutralization assay to prepare monoclonal The monoclonal antibodies were detected by ELISA, the purity of monoclonal antibodies was detected by SDS-PAGE, the concentration of monoclonal antibodies was detected by BCA, the affinity of monoclonal antibodies was detected by Biacore method, the subtype of monoclonal antibodies was identified by subtype reagents, Western blot was performed to test the ability of each monoclonal antibody to cross-neutralize with different types of NOV VLPs-salivary human histopathology (HBGA) and the binding ability of mAbs to different NOV VLPs. Results Three cell lines with neutralizing activity were selected and named as E12, B7 and H7 respectively. The titer of the three monoclonal antibodies were all above 106 with the concentrations of 6.932, 2.662 and 2.371 mg / ml, respectively. The affinity The constants KD are 10-9 mol / L, the purity of the monoclonal antibody E12 is above 93%, belonging to the Ig G2a subtype. The purity of the monoclonal antibodies B7 and H7 are over 98% respectively, belonging to the Ig G1 subtype. Three monoclonal antibodies only had no neutralization activity against GII.17 No V, no neutralization activity against No V of other types, and all of them reacted with eight antigens, but the binding ability of E12 to eight antigens was higher than that of B7 H7 difference. Conclusion The monoclonal antibody against GⅡ.17 No V has been successfully prepared, which lays the foundation for further study on the biological characteristics of the virus and the development of the vaccine.