Fc-沉默型抗人CD36嵌合抗体的制备及作用初步鉴定

Preparation and preliminary identification of Fc-silent anti-human CD36 chimeric antibody

目的制备Fc-沉默型抗人CD36嵌合抗体,分析其生物活性及对CD36(+)血小板的吞噬影响。方法通过杂交瘤细胞株RNA提取、PCR扩增及序列分析获得单克隆抗体(mAb)32-106的V H和V L基因序列;利用基因合成和重组技术构建抗人CD36嵌合抗体轻、重链的表达载体;经Zeocin及杀稻瘟菌素筛选,获得稳定表达嵌合抗体的细胞株;利用亲和层析柱纯化抗体;SDS-PAGE检测嵌合抗体纯度及分子量;ELISA及流式细胞术测定抗体结合CD36抗原的活性;通过血小板吞噬实验分析嵌合抗体参与CD36(+)血小板吞噬的能力。结果成功构建嵌合抗体轻、重链表达载体;共转染HEK293细胞后,经筛选及克隆化获得稳定表达嵌合抗体的细胞株;蛋白银染证实嵌合抗体的纯度高及分子量正确;流式细胞术及ELISA结果表明嵌合抗体具有结合人CD36抗原的活性;血小板吞噬实验表明Fc-沉默型抗人CD36嵌合抗体基本丧失介导单核细胞吞噬CD36(+)血小板的能力。结论本研究成功制备了Fc-沉默型抗人CD36嵌合抗体,并在体外证实其丧失介导CD36(+)血小板清除的能力,为修饰抗体用于CD36抗体介导的胎儿和新生儿同种免疫性血小板减少症(FNAIT)治疗研究奠定了理论基础。

Objective To prepare a Fc-silent chimeric antibody against human CD36 and analyse its bioactivity and phagocytic effect on CD36(+)platelets.Methods The genes encoding V H and V L fragments of the monoclonal antibody(mAb)32-106 were obtained through RNA extraction from hybridoma cell lines,PCR amplification and sequence analysis.By using gene synthesis and recombination techniques,the vectors expressing light and heavy chains of the chimeric antibody against human CD36 were constructed.Stable cell lines secreting the chimeric antibody were established by selective culture using Zeocin and Blasticidin.Antibodies were purified by an affinity chromatography column.The purity and molecular weight of the chimeric antibody were detected by SDS-PAGE.The activity of the antibody binding to CD36 antigen was determined by ELISA and flow cytometry.The ability of the chimeric antibody to participate in CD36(+)platelet phagocytosis was analyzed by a platelet phagocytosis assay.Results The vectors expressing light and heavy chains of chimeric antibody were successfully constructed.After co-transfection into HEK293 cells,stable cell lines secreting chimeric antibody were obtained by screening and cloning.The high purity and correct molecular weight of the chimeric antibody were confirmed by protein silver staining.The results of Flow cytometry and ELISA showed that the chimeric antibody had the activity of binding to human CD36 antigen.Platelet phagocytosis assay showed that the Fc-silent chimeric antibody against human CD36 basically lost the ability of mediating monocyte phagocytosis of CD36(+)platelets.Conclusion In this study,the Fc-silent chimeric antibody against human CD36 was successfully prepared,and its loss of ability to mediate CD36(+)platelet clearance was confirmed in vitro,which provides a preliminary basis for the study of modified antibodies for the therapy of CD36-antibody-mediated fetal and neonatal alloimmune thrombocytopenia(FNAIT).