Gene-modified TIGIT protein, monoclonal antibody and application thereof

A monoclonal antibody, protein technology, applied in the direction of anti-animal/human immunoglobulin, application, genetic engineering, etc., to achieve broad market prospects and high specificity

Active Publication Date: 2020-09-25
江苏莱森生物科技研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no TIGI recombinant protein with glycosylation modification. If a glycosylation-modified anti-TIGI monoclonal antibody can be d

Method used

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  • Gene-modified TIGIT protein, monoclonal antibody and application thereof
  • Gene-modified TIGIT protein, monoclonal antibody and application thereof
  • Gene-modified TIGIT protein, monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Preparation of anti-TIGIT monoclonal antibody

[0027] (1) Expression and purification of recombinant TIGIT protein

[0028] a. Transform Escherichia coli BL21 to express the target recombinant protein

[0029] The Genbank accession number of the TIGIT gene is 201633. The glycosylation modification of the TIGIT gene is carried out. The nucleotide sequence of the modified TIGIT gene is shown in SEQ ID NO. 1. The sequence was synthesized and inserted by Nanjing GenScript Biotechnology Co., Ltd. The prokaryotic expression vector pET-30a constitutes the recombinant expression plasmid pET-30a-TIGIT (glycosylated), and pET-30a-TIGIT (glycosylated) is transformed into E. coli BL21 by heat shock at 42°C for 90 s, and kanamycin is picked Resistant bacterial plaques were cultured in 500 mL LB medium until the OD of the bacterial solution was 0.6–0.8, and then 0.5 mM IPTG was added to induce the expression of the target protein. After incubation at a constant temper...

Embodiment 2

[0038] Example 2: TIGIT monoclonal antibody titer determination and specificity

[0039] (1) Titer determination of monoclonal antibody:

[0040] Dilute the TIGIT protein to 1 μg / mL with pH9.6, 0.05 mol / L carbonate buffer, coat the microplate plate, 100 μL / well, overnight at 4°C; use sample diluent (containing 0.5% bovine serum Phosphate buffered saline solution of albumin) The glycosylated TIGIT monoclonal antibody obtained in Example 1 and the TIGIT monoclonal antibody were respectively mixed at a ratio of 1:10 3 , 1:10 4 , 1:10 5 , 1:10 6 Dilute, 100 μL / well, incubate at 37°C for 1 h; take out the microplate plate, wash it 3 times with TBS-T, pat dry the microplate plate, add 100 μL 1:5000 diluted HRP-labeled goat anti-mouse II to each well Antibody, incubated at 37°C for 30 min; after washing 5 times with TBS-T, add 100 μL of TMB substrate display solution to each well, develop color at 37°C in the dark for 10-15 min, then add 50 μL of stop solution to stop the reactio...

Embodiment 3

[0043] Embodiment 3: Western blot experiment of TIGIT monoclonal antibody

[0044] Collect human CD8+ T cells (purchased from ATCC cell bank in the United States) and human CD4+ T cells (purchased from ATCC cell bank in the United States), lyse the cells with RIPA lysate, centrifuge at 5000 g 4°C for 20 min, collect the supernatant, and use BCA reagent Cell lysate was separated by 12% SDS-PAGE electrophoresis, and then the PAGE gel (purchased from Beyond Biotechnology Co., Ltd.) was placed on PVDF membrane, and transfected at 120 mA. Membrane for 240 min; put the PVDF membrane into 5% skimmed milk and block at room temperature for 1 h, and then use the 1:5000 glycosylated TIGIT monoclonal antibody provided in the present invention as the primary antibody to incubate overnight at 4°C; take out the PVDF membrane, PBS- Rinse 3 times at T, 5 min each time, place in 1:5000 diluted HRP-labeled goat anti-mouse secondary antibody and incubate at room temperature for 1 h, discard the s...

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Abstract

The invention relates to the technical field of biology, in particular to a gene-modified TIGIT protein, a monoclonal antibody thereof and application of the gene-modified TIGIT protein to glycosylation modification of TIGIT to obtain an optimized glycosylated TIGIT protein, and a murine monoclonal antibody is prepared by immunization of the protein. The anti-TIGIT monoclonal antibody obtained bythe invention has the characteristics of high specificity and mass production, and the titer of the obtained antibody reaches 1*10<5> or above. The anti-TIGIT monoclonal antibody obtained by the invention can be specifically combined with TIGIT protein, can be used for immunological detection of cells, has a wide market prospect, and has important clinical significance for further developing biological treatment drugs taking TIGIT as a target.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gene-modified TIGIT protein and its monoclonal antibody and application. Background technique [0002] Tumor immunotherapy is a research hotspot in the field of tumor treatment in recent years. Both innate and adaptive immune systems can respond to tumors. In the stage of tumor immune surveillance, a variety of cells participate in this process, such as NK cells, T cells and macrophages. Cells, etc. respond through cellular immunity, and regulate anti-tumor effects by secreting cytokines. T cell immunoglobulin domain and immunoreceptor tyrosine inhibitory motif (TIGIT) is an immunomodulatory molecule, mainly expressed on the surface of T cells, NK cells and regulatory T cells, etc. Shared inhibitory receptors. In cancer cells, the expression of TIGIT was significantly up-regulated, which inhibited NK cells, CD8 + T cells, CD4 + Activation of T cells and regulatory T cells, ther...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N5/20C07K14/705C07K16/28G01N33/68G01N33/577C12R1/91
CPCC07K14/70503C07K16/2803G01N33/6872G01N33/577C07K2317/33Y02A50/30
Inventor 刘晗青屠志刚
Owner 江苏莱森生物科技研究院有限公司
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