特定激活性吞噬受体的配体与广谱肿瘤抗原的化学生物学桥接  

Biochemical Bridging of Ligand for Specific Activating Phagocytic Receptor and Broad-Spectrum Tumor Antigens

作　　者：张国秀 郑晨晨 赵智辉[1] Zhang Guoxiu;Zheng Chenchen;Zhao Zhihui(School of Life Sciences,Nanjing Normal University,Institute of Biochemistry and Biological Products,Jiangsu Key Laboratory for Molecular and Medical Biotechnology,Nanjing 210023,China)

机构地区：[1]南京师范大学生命科学学院,生物化学与生物制品研究所,江苏省分子与医学生物技术重点实验室,江苏南京210023

出　　处：《南京师大学报（自然科学版）》2020年第2期92-99,共8页Journal of Nanjing Normal University(Natural Science Edition)

基　　金：国家自然科学基金重大研究计划集成项目子课题(2013104GZ90073).

摘　　要：目前肿瘤疫苗制备方法尚无法做到将广谱肿瘤抗原通过特定激活性吞噬受体途径递送给抗原呈递细胞,这可能是现有肿瘤疫苗临床疗效不佳的原因之一.因此,本研究利用化学生物学方法,尝试将激活性吞噬受体的配体与广谱肿瘤抗原连接,为制备肿瘤疫苗提供新的方法.首先,通过非天然糖代谢掺入对培养的小鼠4T1乳腺癌细胞和CT26.WT结直肠癌细胞抗原进行标记,使糖基化肿瘤抗原携带叠氮化的唾液酸,利用流式细胞术和免疫荧光技术确定代谢掺入的最优条件参数.其次,利用生物正交反应使肿瘤抗原叠氮修饰位点再进一步共价缀合生物素,利用Western Blot检测蛋白的生物素化情况.最后,利用抗原-抗体结合原理,将小鼠抗生物素单抗(IgG1亚型,其Fc结构域是IgG1FcR的配体)与生物素化肿瘤抗原交联形成免疫复合物,通过Western Blot判断最优交联参数.实验结果表明,唾液酸前体物代谢掺入能高效地使肿瘤细胞发生叠氮化修饰,最佳浓度是2 mmol/L、最佳时间是24 h;通过生物正交反应能使叠氮化肿瘤抗原进一步生物素化;生物素化肿瘤抗原能高效地与抗生物素抗体形成复合物,二者交联的最佳比例(w/w)是20/1(4T1)或25/1(CT26.WT).结果证明,本研究通过化学生物学手段实现了广谱肿瘤抗原与特定吞噬受体的配体的交联,在方法学上为研制新型肿瘤疫苗奠定了基础.The current methods for preparing tumor vaccines has not been able to deliver broad-spectrum tumor antigens(TAgs)to antigen-presenting cells(APCs)through specific activating phagocytic receptor(SAPR)pathways,which may be one of the reasons for the poor clinical efficacy of existing tumor vaccines. Here,by using biochemical methods,it was tried to achieve the linkage of SAPR ligand with broad spectrum TAgs,attempting to provide a novel strategy for the preparation of tumor vaccines. Firstly,the antigens of 4 T1 breast cancer cells or CT26.WT colorectal cancer cells were decorated by metabolic incorporation(MI)of non-natural carbohydrates,so that glycosylated tumor antigens could carry azide group on sialic acid residue,the optimal parameters of MI were determined by flow cytometry and immunofluorescent assay. Secondly,the azide modified sites of TAgs were further covalently conjugated with biotin through bio-orthogonal reaction,and the biotinylated tumor antigens(bio-TAgs)were detected by Western Blot. Finally,based on the principle of antigen-antibody binding,mouse anti-Biotin monoclonal antibody(anti-Biotin mAb,IgG1 subtype,whose Fc domain is a ligand of IgG1 FcR)was cross-linked with bio-TAgs to form immune complexes,and the optimal cross-linking parameters was determined by Western Blot. The results showed that TAgs were efficiently modified by azide through MI,the optimal cultural concentration of sialic acid precursor is 2 mmol/L,and the optimal time of MI is 24 h. The azide labeled TAgs can further become biotinylated via bio-orthogonal reaction,the bio-TAgs can efficiently cross-linked with the anti-Biotin mAb to form immune complexes,and the optimal ratio(w/w)of anti-Biotin mAb and bio-TAgs for cross-linkage are 20/1(4 T1)or 25/1(CT26.WT). It is concluded that broad-spectrum tumor antigens can cross-link with SAPR ligand by biochemical methods,which lays a foundation for the development of new tumor vaccines.

关 键 词：代谢掺入 生物正交反应 激活性吞噬受体 抗原递送 肿瘤疫苗 

分 类 号：R730.3[医药卫生—肿瘤]