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Anti-LAG3 protein monoclonal antibody and cell strain, preparation method and application thereof

A monoclonal antibody and protein technology, applied in the field of biomedical engineering, can solve the problem of unclear signal transduction mechanism

Active Publication Date: 2021-08-10
FUZHOU MAIXIN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

MHC class II molecules are the ligands of LAG3. Compared with CD4, LAG3 has a higher affinity with MHC class II molecules, and secretes IL-10, TGF-β and other negative agents by binding to MHC class II molecules on the surface of antigen-presenting cells. Sexual cytokines inhibit the cytotoxicity of T cells, but the specific signal transduction mechanism in the cells is still unclear

Method used

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  • Anti-LAG3 protein monoclonal antibody and cell strain, preparation method and application thereof
  • Anti-LAG3 protein monoclonal antibody and cell strain, preparation method and application thereof
  • Anti-LAG3 protein monoclonal antibody and cell strain, preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0018] The preparation of embodiment 1 immunogen

[0019] 1. Immunogen preparation

[0020] According to the sequence and secondary structure analysis of the protein sequence with the accession number P18627 in Uniprot, the molecular weight of the LAG3 protein with a total length of 260 amino acids is about 39 kDa. According to the predicted protein secondary structure (secondary structure) and surface accessibility (Surface Accessibility) parameters through the online server http: / / www.cbs.dtu.dk / services / NetSurfP / , and through its antigenicity index ( Jameson BA,WolfH.The antigenic index:a novel algorithm for predicting antigenic determinants.Comput Appl Biosci.1988,4(1):181-6.) analysis results, select the 32-51 amino acid sequence SEQ ID NO.1 It is chemically synthesized as an antigenic peptide. To facilitate coupling, a cysteine ​​is added to the carboxyl terminus of the polypeptide to provide sulfhydryl coupling.

[0021] 2. Coupling and purification of peptides

[00...

Embodiment 2

[0023] The establishment of embodiment 2 hybridoma cell lines

[0024] 1. Immunity

[0025] The cross-linked polypeptide in Example 1 was emulsified with complete Freund's adjuvant (Sigma, F5881), immunized with ICR mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and injected subcutaneously into each mouse for 6 Point, the dose is 60μg / only. Immunization was boosted once every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma, F5506) at a dose of 30 μg per mouse. 7 days after the third booster immunization, indirect ELISA (wavelength 450nm) was used to detect the polyantibody titer of the anti-immunogen in the mouse serum. 50μg / only.

[0026] 2. Cell Fusion

[0027] Aseptically prepare the mouse splenocyte suspension that reaches the immune standard, mix it with mouse myeloma cell sp2 / 0 (ATCCNumberCRL-8287) at a ratio of 5:1, and centrifuge at 1500rpm for 5min. After the supernatant was discarded, the ce...

Embodiment 3

[0030] Example 3 Preparation of Monoclonal Antibody by Ascites Induction Method

[0031] 1. Ascites preparation

[0032] The cells in the logarithmic growth phase were washed with serum-free medium and suspended, counted about 5×10 5 , 1ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The removed ascites was centrifuged at 4000rpm for 10min at 4°C. Carefully suck out the ascites in the middle and collect in a centrifuge tube, and store at 4°C or -20°C.

[0033] 2. Purification of monoclonal antibodies

[0034] Antibody was purified from ascitic fluid by HiTrap rProtein A FF (GE Company) affinity chromatography according to the instructions. The purity was identified by SDS-PAGE gel, and the concentration was determined by Bradford method. Purified antibodies were stored at -20°C.

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Abstract

The invention relates to a monoclonal antibody capable of recognizing a human LAG3 antigen, a secretory cell strain, a preparation method of the monoclonal antibody and application of the secretory cell strain in immunodetection. According to the technical scheme, amino acids from the 32nd site to the 51st site at the C tail end of the LAG3 protein are selected as antigen peptides, an immunogen obtained after coupling of KLH is used for immunizing a mouse, and the mouse hybridoma cell strain 22G3 capable of efficiently secreting the anti-LAG3 protein monoclonal antibody and the anti-LAG3 protein monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing LAG3 protein, and is suitable for immunological detection, especially immunohistochemical detection.

Description

technical field [0001] The invention relates to the field of biomedical engineering, in particular to an anti-LAG3 protein monoclonal antibody and its cell line, preparation method and application. Background technique [0002] Lymphocyte-activation gene-3 (LAG3) is an important class of immunosuppressive molecules. LAG3 is regarded as the third immune checkpoint after PD-1 and CTLA-4. It was first introduced in 1990 Discovered by Triebel and colleagues. LAG3 is a type I transmembrane protein containing 498 amino acids on the surface of human activated NK cells and T cells. The LAG3 gene is located on human chromosome 12 and is adjacent to the CD4 gene. Both have the same exons and Intron, so LAG3 has high homology with CD4 molecule. MHC class II molecules are the ligands of LAG3. Compared with CD4, LAG3 has a higher affinity with MHC class II molecules, and secretes IL-10, TGF-β and other negative agents by binding to MHC class II molecules on the surface of antigen-prese...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N5/20G01N33/574G01N33/577
CPCC07K16/2803G01N33/57484G01N33/57407G01N33/577C07K2317/56C07K2317/92
Inventor 杨清海王小亚周洪辉陈惠玲程本亮
Owner FUZHOU MAIXIN BIOTECH CO LTD
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