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TIM-3 nanobody as well as preparation method and application thereof

A technology of TIM-3 and nano-antibody, applied in biochemical equipment and methods, antibody, DNA preparation, etc., can solve the problems of high storage cost, high price of monoclonal antibody drugs, difficulty in large-scale production of monoclonal antibody, etc. achieve high activity

Active Publication Date: 2020-06-09
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mouse monoclonal antibody, the application of human body will produce anti-mouse monoclonal antibody, it can not be used repeatedly, affecting its curative effect
[0008] (2) Existing monoclonal antibodies are relatively large in size and cannot enter tumor tissues well; the development cycle of monoclonal antibodies is long, the production cost is high, and the output is low; monoclonal antibody drugs are expensive, complex in research and development, and humanized Complicated, with limited success
[0009] (3) Existing monoclonal antibodies are difficult to produce on a large scale, and monoclonal antibody drugs consume a huge amount of money in the process of building factories and producing them
[0010] (4) The existing monoclonal antibodies are unstable, easy to degrade, and have high storage costs; easy to contaminate, and high maintenance costs
It has the same structure as the VH of a human antibody, and sequencing shows that it has a high homology with VH3, but the CDR1 (Complementarity-determining region-1) and CDR3 (Complementarity-determining region-3) of the nanobody are relatively long

Method used

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  • TIM-3 nanobody as well as preparation method and application thereof
  • TIM-3 nanobody as well as preparation method and application thereof
  • TIM-3 nanobody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] The vector construction method provided by the embodiment of the present invention is:

[0116] 1. Target fragment amplification is specifically:

[0117] (1) Design synthetic primers to amplify a sufficient amount of the target product by PCR.

[0118] (2) The PCR reaction uses pfu high-temperature polymerase.

[0119] The consumption of each component of PCR: (primer concentration is that 10D is dissolved in 400ul ddH 2 O)

[0120] Reaction system: 50ul. Primer mix, (1 / 28) 0.4ul x 13.6 total 8ul. 10X pfu Buffer, 5ul. Each upstream and downstream primers, each 2ul. Pfu, 0.4ul (5u / ul). wxya 2 O, replenish water to 50ul respectively.

[0121] The method amplification specific steps of target fragment PCR are:

[0122] (1) The first round of PCR procedure:

[0123]

[0124] The above is the first-round PCR reaction system, and the second-round PCR is performed with the first-round PCR product as a template.

[0125] (2) The second round of PCR reaction syst...

Embodiment 2

[0140] The construction of the nanobody library provided by the embodiments of the present invention is specifically:

[0141] 1) Experimental design

[0142] The M13 phage display system was selected to display the VHH antibody library, which consists of pMECS phagemid vector, E.coliTG1 and M13KO7 helper phage. In the phagemid vector pMECS, the sequence before the Pst I restriction site is the coding sequence of the pelB secretion signal peptide and some amino acids in the first framework region of the antibody. The pelB signal peptide can guide the secretion of subsequent polypeptides into the periplasmic cavity. The Not I restriction site is followed by the coding sequence of HA and 6×His tag, which can be used for purification or detection of fusion protein. The sequence immediately following it encodes the phage PIII capsid protein ( Figure 4 shown). There is an amber stop codon between the 6×His tag and the gene III sequence, and 10% to 20% of the amber stop codon ca...

Embodiment 3

[0161] The identification and expression of proteins provided in the embodiments of the present invention are specifically:

[0162] (1) Construction of mammalian cell expression vector (plasmid template, His-Flag tag added to the C-terminus)

[0163] 1. Amplify and extract the vector plasmid containing the target gene.

[0164] 2. Subcloning into eukaryotic expression vector pcDNA3.1.

[0165] 3. Sequencing to verify the accuracy of the constructed plasmid.

[0166] 4. Obtain the recombinant plasmid pcDNA3.1 by pumping.

[0167] (2) Mammalian cell culture, protein expression and purification small test

[0168] 1. Cell lines and materials

[0169] Cell line: HEK293 cells.

[0170] Medium: DMEM (10% serum), DMEM (serum-free).

[0171] Petri dish: 10cm dish or 15cm dish

[0172] 2. HEK293 cell transfection (10cm dish)

[0173] (1) 24 hours before transfection with 4-5X10 6 The total amount of cells per 10cm culture dish is plated, and the transfection can be carried ou...

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Abstract

The invention belongs to the technical field of resistant substances from animals or humans, and discloses a TIM-3 nanobody as well as a preparation method and application thereof. The nanobody is TIM-3 nanobody, and the sequence is shown in SEQ ID NO:1; and a TIM-3 antigen is expressed by mammalian cell transient transfection, a nanobody library is screened for multiple times by using the TIM-3 antigen to obtain specific nanobody phage, and sequencing is performed to obtain a target fragment. An HEK293 cell line is used to express the antigen, a mammalian expression system is used to expresshuman protein to guarantee the original structure of the protein to the maximum extent to ensure that the protein has post-translational modification and specific modification, such as glycosylation of eukaryotic protein, so that the obtained protein has higher activity; the method guarantees the original structure and activity of the protein to the greatest extent; and the screened nanobody can efficiently and specifically bind to a target.

Description

technical field [0001] The invention belongs to the technical field of immune substances from animals or humans, and in particular relates to a TIM-3 nanobody, a preparation method and an application thereof. Background technique [0002] At present, T-cell immunoglobulin and mucin-3 (T-cell immunoglobulin and mucin-3, TIM-3), a membrane protein, is a new member of the T-cell immunoglobulin family. It was first found in Th1 lymphocytes and CD8+ T lymphocytes. The mouse TIM-3 coding gene exists on chromosome 11, which has a high degree of similarity with human TIM-3, while the human TIM-3 coding gene exists on chromosome 5 and contains 7 exogenous Exon. [0003] Studies suggest that TIM-3 is only selectively expressed on activated Th1 cells and can be used as a new marker to distinguish Th2 cells from Th2 cells. TIM-3 can exert the function of inhibiting Th1 cells by combining with its ligand. It participates in the occurrence of inflammation, can regulate the activation ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/85C12N15/10A61K39/395A61P35/00
CPCC07K16/2803C12N15/85C12N15/1037A61P35/00C07K2317/569C07K2317/22C07K2317/76C07K2317/92C12N2800/107A61K2039/505Y02A50/30
Inventor 陈创夫吴鹏
Owner SHIHEZI UNIVERSITY
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