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《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

Issue:

2020年02期

Page:

92-99

Research Field:

·生物学·

Publishing date:

Info

Title:

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

Author(s):

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

Keywords:

metabolic incorporation; bio-orthogonal reaction; activating phagocytic receptor; antigen delivery; tumor vaccine

PACS:

R730.3

DOI:

10.3969/j.issn.1001-4616.2020.02.015

Abstract:

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 4T1 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(4T1)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.

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