CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) nanoantibody and preparation method and application thereof

A CTLA-4, 1. CTLA-4 technology, applied in the field of biomedicine, can solve the problems of easy degradation, long development cycle of monoclonal antibodies, and expensive monoclonal antibody drugs, achieve high activity, ensure the original structure and active effect

Inactive Publication Date: 2019-09-20
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the monoclonal antibody is used for mouse monoclonal antibody, the application to the human body will produce anti-mouse monoclonal antibody, which cannot be used repeatedly, which will affect its curative effect
[0005] (2) The monoclonal antibody is relatively large in size and cannot enter the tumor tissue well
[0006] (3) The monoclonal antibody development cycle is long, the production cost is high, and the output is low
[0007] (4) Monoclonal antibody drugs are expensive, complex in research and development, complex in humanization, and limited in success rate
[0008] (5) It is 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
[0009] (6) Unstable, easy to degrade, high storage cost; easy to pollute, high maintenance cost
It has the same structure as the VH of human antibodies, 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 Nanobodies are relatively long

Method used

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  • CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) nanoantibody and preparation method and application thereof
  • CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) nanoantibody and preparation method and application thereof
  • CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) nanoantibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1 CTLA-4 nanobody, preparation method

[0054] S1: Synthesize and amplify CTLA-4 immune antigen sequence fragments.

[0055] First, the CTLA-4 immune antigen sequence was obtained through the Uniprot database, see SEQ ID NO:1 for details. Then, use the Primer Premier 5.0 software to design CTLA-4 upstream primers and CTLA-4 downstream primers, as shown in the table below and SEQ ID NO:2 and SEQ ID NO:3.

[0056]

[0057] The above primers and antigen sequences were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis, and the synthesized fragments were used as PCR templates for amplification.

[0058] The PCR system is: (primer concentration is 1OD dissolved in 400ul ddH 2 O)

[0059]

[0060] PCR program:

[0061]

[0062] After the completion of PCR, 1% agarose gel electrophoresis was performed, and the fragments were recovered according to the operation of the recovery kit.

[0063] PCR results such as figure 2 As shown, the tar...

Embodiment 2

[0185] Example 2 Application of CTLA-4 nanobody

[0186] Cell experiment: Carry out preliminary in vitro cell pharmacodynamic evaluation on CTLA-4 nanobody to verify its effectiveness. The nanobody screened by the present invention is used for lethality experiments on MC-38:C57 / BL6 mouse colon cancer cells.

[0187] In the present embodiment, the cellular immunofluorescence method (semi-quantitative) is specifically adopted, and the specific scheme is as follows:

[0188] 1. When MC-38 cells grow confluent to 95%-100% on coverslips, take them out from the incubator.

[0189] 2. Wash 3 times with pre-warmed 1×PBS, 10 minutes each time.

[0190] 3. Fix with 4% formaldehyde at room temperature for 20-30 minutes.

[0191] 4. Wash 3 times with 1×PBS, 10 minutes each time.

[0192] 5. Permeabilize with 0.2% Triton X-100 for 2-5 minutes.

[0193] 6. Wash 3 times with 1×PBS, 10 minutes each time.

[0194] 7. Block with 5% BSA at room temperature for 30 minutes.

[0195] 8. Add ...

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Abstract

The invention belongs to the technical field of biomedicine and particularly relates to a CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) nanoantibody and a preparation method and application thereof. The ability to bind with antigens can be improved or collaboratively enhanced through the CTLA-4 antibody. The CTLA-4 antibody has no complete antibody structure and lacks Fc end and Y-shaped structure; the antigens bound to the CTLA-4 antibody are not easily recognizable and can escape easily from being caught by the immune system. The technical means includes mainly: carrying out antigen-vector connecting; transferring to competent cells, and extracting recombinant plasmids; transfecting to host bacteria for expressing; immunizing camels with an antigen protein; collecting peripheral blood, extracting total RNA of a lymphocyte sample, inversely transcribing the total RNA into cDNA, performing amplifying to obtain a VHH library fragment, inserting the VHH library fragment into an expression vector, and transferring to the competent cells to construct an antibody immunization library; screening nanoantibodies in the antibody immunization library; verifying binding of the screened nanoantibodies with a CTLA-4 antigen.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a CTLA-4 nanometer antibody, a preparation method and an application thereof. Background technique [0002] Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a T lymphocyte transmembrane protein, which was first discovered when screening the cDNA library of mouse killer T cells. CTLA-4 has a high degree of endocytosis, and usually exists in intracellular bodies in the form of dimers, and its conformation will not be changed when combined with its ligand. CTLA-4 can bind to the B7 receptor on the surface of antigen-presenting cells or activated T cells, and inhibit the function of cells through reverse transduction to signal. The study found that high-mobility group box protein B1 can significantly reduce the expression level of T lymphocyte toxicity-associated antigen-4 expressed by mouse regulatory T cells, and pretreatment with blocked TLR-4, and then tre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00
CPCC07K16/2818A61P35/00C07K2317/569C07K2317/22C07K2317/73C07K16/2803C07K2317/70C07K2317/76C12N15/1037C40B40/02C12Q1/686C12Q1/6886
Inventor 陈创夫吴鹏
Owner SHIHEZI UNIVERSITY
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