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Specific anti-mouse TIGIT monoclonal antibody and preparation method, identification and application thereof

A monoclonal antibody and mouse technology, applied in biochemical equipment and methods, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-animal/human immunoglobulin, etc., can solve tumor immunity Tolerance, tumor immune escape and other issues, to achieve the effect of good specificity and high titer

Active Publication Date: 2013-05-01
IMMUNOPHARMACEUTIC INST OF HEFEI RUIDA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In melanoma-bearing mice, the expression of TIGIT is up-regulated, and the tumor cells highly express the ligand PVR, and the ability of TIGIT to bind PVR is much higher than that of CD226 / CD96, which inhibits the activity of NK cells and causes tumor immune tolerance. tumor immune escape

Method used

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  • Specific anti-mouse TIGIT monoclonal antibody and preparation method, identification and application thereof
  • Specific anti-mouse TIGIT monoclonal antibody and preparation method, identification and application thereof
  • Specific anti-mouse TIGIT monoclonal antibody and preparation method, identification and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Construction of eukaryotic expression vector pcDNA3-mTIGIT-hFc

[0059] From the pMD18-T-full-length mouse TIGIT vector (pMD18-T was purchased from Takara, Code: D103A), the coding sequence of the mouse TIGIT extracellular segment was amplified by PCR, and the sequence fragment was combined with the pcDNA3-hFc recombinant plasmid (pcDNA3 was purchased from Invitrogen , No. V790-20) were ligated after enzyme digestion at EcoRI and Xho I, and the ligated product was transformed into DH5α Escherichia coli, plated and grown overnight, and the screened strains were subjected to PCR detection, and DNA sequencing was performed on the positive clones identified by PCR. The sequencing results showed that the vector pcDNA3-mTIGIT-hFc was constructed successfully. The construction process of the eukaryotic expression vector pcDNA3-mTIGIT-hFc is as follows: figure 1 shown.

Embodiment 2

[0060] Example 2: Expression, purification and identification of fusion protein mTIGIT-hFc

[0061] 1. Expression of fusion protein mTIGIT-hFc (SEQ ID NO: 3)

[0062] Transfect the pcDNA3-mTIGIT-hFc vector into 293A cells (purchased from Shanghai Cell Bank, catalog number: GNHu 1), collect the serum-free culture supernatant, concentrate the supernatant with a 30kD membrane (purchased from Biyuntian), and use Protein G to purify the column (purchased from GE Healthcare, HiTrap Protein G HP, catalog number: 1700404-01) enrich the target protein on the concentrated supernatant, then use acidic PBS (pH2.8) to elute, and then use alkaline neutralized Tris buffer (pH9.0) to obtain mTIGIT-hFc protein solution. The result is as figure 2 As shown in A.

[0063] 2. Identification of Fusion Proteins

[0064] The purified mTIGIT-hFc protein was identified by SDS-PAGE&Western Blot; the results are as follows figure 2 b.

Embodiment 3

[0065] Embodiment 3: Preparation of monoclonal antibody

[0066] 1. Preparation of Splenocytes

[0067] The purified mTIGIT-hFc protein was mixed with an equal volume of complete Freund's adjuvant (CFA, purchased from Sigma, product number F5881-10ML), the final concentration of mTIGIT-hFc was 100 μg / ml, the mixture was fully mixed to form water-in-oil, and Rats (purchased from Shanghai Slack, male, 8 weeks old) were immunized for the first time, each rat was injected with 40-60 μg mTIGIT-hFc protein (immunization dose was 133-200 μg / kg rat body weight), and immunized once every 2 weeks . After 5 times of immunization, the enzyme-linked immunosorbent assay (ELISA) detected that the serum titer of the rat was very high. After the rat was sacrificed, the spleen of the rat was taken, passed through a 200-mesh cell sieve, and the filtered spleen cells were collected and left to stand for several minutes. Afterwards, the upper splenocytes were aspirated.

[0068] The method for ...

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Abstract

The invention relates to an anti-mouse TIGIT (T cell Ig and ITIM domain), and a hybridoma cell strain mTIGIT-mAb-13G6 (the preservation NO: CCTCC NO: C201299) for producing the monoclonal antibody, and further relates to a preparation method and the application of the monoclonal antibody. The monoclonal antibody can efficiently prevent combination of mTIGIT and mPVR (poliovirus receptor), and can be used for Western blot, ELISA and Flow Cytometry for mTIGIT molecule detection.

Description

technical field [0001] The invention relates to a monoclonal antibody against mouse TIGIT (full name T cell Ig and ITIM domain). Specifically, it relates to a monoclonal antibody specific to TIGIT, and the present invention also relates to a hybridoma cell line producing the monoclonal antibody, as well as a preparation method and application of the monoclonal antibody. Background technique [0002] Natural killer cells (ie, NK cells), as the main natural immune cells, play an important role in killing virus-infected cells and tumor cells in the body. NK cells are controlled by different activating receptors and inhibitory receptors on their surface NK cell activation, proliferation and killing functions. [0003] TIGIT (T cell Ig and ITIM domain) molecules are newly discovered inhibitory receptors expressed on the surface of NK cells in 2009, mainly including extracellular IgV-like domains, transmembrane regions and intracellular ITIM motifs (Immunoreceptor tyrosine-based ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N5/20G01N33/68G01N33/577
Inventor 孙汭毕嘉成张清郑晓东田志刚魏海明
Owner IMMUNOPHARMACEUTIC INST OF HEFEI RUIDA CO LTD
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