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Monoclonal antibody blocking human tim-3 function and its coding gene and application

A monoclonal antibody, amino acid technology, applied in the direction of antibody, application, genetic engineering, etc., can solve the problem of low affinity of Tim-3 antibody, and achieve the effect of inhibiting signal transduction

Active Publication Date: 2020-11-03
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, the existing Tim-3 antibody has a low affinity, and there is still a lack of a high-affinity blocking Tim-3 antibody

Method used

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  • Monoclonal antibody blocking human tim-3 function and its coding gene and application
  • Monoclonal antibody blocking human tim-3 function and its coding gene and application
  • Monoclonal antibody blocking human tim-3 function and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 Construction of anti-human Tim-3 monoclonal antibody CDT1 hybridoma cell line

[0047] 1. Materials

[0048] DNA immune adjuvant was purchased from Sigma; fetal bovine serum and PRMI1640 medium were purchased from Gibco; SP2 / 0 cells were purchased from ATCC; BALB / c mice were provided by the Experimental Animal Center of Zhejiang University.

[0049] 2. Method:

[0050] (1) Plasmid construction

[0051] Primers were designed according to the sequence of the Tim-3 gene found on NCBI, and the DNA sequence of the Tim-3 gene was amplified using human cDNA as a template, which was constructed on the pcDNA3.1 plasmid using molecular biology methods.

[0052] (2) Immunization of mice

[0053] BALB / c mice aged 4-6 weeks were selected, and the constructed Tim-3 gene recombinant plasmid was dissolved in PBS and an equal volume of adjuvant, mixed and emulsified, and subcutaneously and intraperitoneally multi-point immunized. Every 2 weeks after the first immunizati...

Embodiment 2

[0060] Example 2: Anti-human Tim-3 monoclonal antibody CDT1 antibody subclass identification and stability test

[0061] According to the instructions of the antibody subclass identification kit, the subclass of the antibody was identified, and the result was that the heavy chain was IgG2a, and the light chain was Kappa.

[0062] The hybridoma cell line was continuously subcultured in vitro for 3 months, and the antibody titer in the supernatant was determined; the frozen hybridoma cell line was recovered after 4 months, and the antibody titer in the supernatant was detected, and no significant changes occurred. Changes, indicating that the obtained antibody-producing hybridoma cell line has stable performance.

Embodiment 3

[0063] Example 3: Cloning of heavy and light chains of anti-human Tim-3 monoclonal antibody CDT1

[0064] 1. Materials

[0065] Monoclonal antibody heavy chain and light chain gene amplification primers were designed according to the literature and synthesized at Shanghai Sangon Bioengineering Co., Ltd.; DNA fragment purification kit and plasmid extraction kit were purchased from QIAGEN; pMD-18T kit and reverse transcriptase Purchased from TAKARA Company; Competent cells were purchased from Promega Company; KOD plus fidelity PCR enzyme was purchased from TOYOBO Company; Trizol reagent was purchased from Invitrogen Company.

[0066] 2. Methods and results

[0067] (1) Take 5×10 6 -10 7 The monoclonal antibody hybridoma cells were centrifuged to remove the supernatant, and 1ml Trizol reagent was added to repeatedly pipette until the cells were fully lysed. After standing at room temperature for 5 minutes, add 0.2ml of chloroform, mix up and down, and let stand at room temper...

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Abstract

The invention belongs to the technical field of bioengineering, and specifically relates to a monoclonal antibody capable of blocking human Tim-3 function and a coding gene of the monoclonal antibody.The antibody provided by the invention can bind to the extracellular region of human Tim-3, can specifically block binding of the Tim-3 and a ligand of the Tim-3, and inhibits signal transduction, sothat the monoclonal antibody provided by the invention can be used as a blocking agent of a Tim-3 signal channel, and thus becomes a novel antibody medicine of tumor immunotherapy, and chronic viralinfectious disease and autoimmune disease treatment.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a monoclonal antibody blocking the function of human Tim-3, its coding gene and application. Background technique [0002] McIntire et al. discovered the Tim gene family in 2001, and found that the Tim gene encodes protein molecules containing immunoglobulin and mucin domains, is widely expressed on immune cells, and participates in regulating the differentiation of various T cells, including regulatory T cells. Play an important role in immune homeostasis and clearance of apoptotic cells. The Tim family has only three genes in humans, which are located at 5q33.2 of the human chromosome and encode proteins Tim-1, 3, and 4, respectively. Tim-3 is an important member of the Tim family, consisting of 301 amino acids, and its structure includes extracellular immunoglobulin V region, mucin region, transmembrane region and cytoplasmic region [FreemanGJ, Casasnovas J...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00A61P37/00A61P31/04
CPCA61K2039/505C07K16/2803C07K2317/565
Inventor 陈智朱海红任艳丽楼国华郑敏
Owner ZHEJIANG UNIV
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