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Anti-PD-1 nano antibody, coding gene, recombinant nano antibody, recombinant vector, recombinant strain and application

A nanobody, PD-1 technology, applied in the field of biomedicine, can solve the problems of strong immunogenicity, high cost and weak tissue penetration

Active Publication Date: 2022-04-26
NINGXIA MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above monoclonal antibodies have the defects of weak tissue permeability, strong immunogenicity and high cost.

Method used

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  • Anti-PD-1 nano antibody, coding gene, recombinant nano antibody, recombinant vector, recombinant strain and application
  • Anti-PD-1 nano antibody, coding gene, recombinant nano antibody, recombinant vector, recombinant strain and application
  • Anti-PD-1 nano antibody, coding gene, recombinant nano antibody, recombinant vector, recombinant strain and application

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preparation example Construction

[0040] The preparation method of the fusion gene is not particularly limited in the present invention, and it can be prepared by artificial synthesis. In the embodiment, based on the PAS (PCR-based Accurate Synthesis) method, a full-length splicing primer is designed, and at both ends of the primer Protective base synthesis target genes were designed for each.

[0041] The present invention also provides a recombinant expression vector comprising the above-mentioned fusion gene, and the basic vector of the recombinant expression vector includes a prokaryotic expression vector.

[0042] The prokaryotic expression vector of the present invention preferably includes pCZN1. In the embodiment of the present invention, preferably, the nucleotide sequence shown in the above SEQ ID No. 5 is inserted between the NdeI and XbaI sites of the pCZN1 plasmid vector. The method for preparing the recombinant expression vector is not particularly limited in the present invention, and conventio...

Embodiment 1

[0051] For the construction of natural camel-derived nanobody gene library:

[0052] (1) Total RNA from camel spleen tissue was extracted by Trizol method, purified using the RNA purification kit provided by TIANGEN, and cDNA was obtained by reverse transcription according to the Thermo Scientific ReverAid First Strand cDNA Synthesis Kits kit.

[0053] (2) Using cDNA as a template, amplify the variable region fragment of the heavy chain antibody by nested PCR;

[0054] ①The first round of PCR:

[0055] Upstream primer: 5'-GTCCTGGCTGCTCTTTCTACAAAG-3' (SEQ ID No.20)

[0056] Downstream primer: 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID No.21)

[0057] 20μl reaction system for the first round of PCR: cDNA 2μl, Mix 10μl, upstream primer 1μl, downstream primer 1μl and the rest ddH 2 O.

[0058] The reaction conditions of the first round of PCR amplification were: 95°C for 5min; 32 cycles of 95°C for 30s, 55°C for 30s, 72°C for 45s; 72°C for 10min.

[0059] Amplified Heavy Chain Ant...

Embodiment 2

[0085] Expression and purification of recombinant nanobodies in host bacteria Escherichia coli:

[0086] (1) Connect the Nanobody sequence (SEQ ID No.1) obtained by the sequencing analysis to the human IgG Fc segment and subclone it into the pCZN1 plasmid vector, and transform it into Escherichia coli Arctic Express. The clones were inoculated in a test tube containing 50 μg / mL Amp in 3 mL of LB culture medium, and shaken at 220 rpm at 37°C overnight; (2) the next day, inoculated in 30 mL of LB culture medium with 50 μg / mL Amp at a ratio of 1:100, and shaken at 220 rpm at 37°C. Shake to cell OD 600 0.6 to 0.8, add IPTG to a final concentration of 0.5mM, shake overnight at 20°C and 220rpm to induce expression of the fusion protein; (3) Collect the bacteria and perform ultrasonic crushing to obtain a bold liquid of the inclusion body protein, which is then passed through a Ni column for affinity Purify the fusion protein. Figure 4 It is the purified recombinant nanobody, and ...

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Abstract

The invention provides an anti-PD-1 nano antibody, a coding gene, a recombinant nano antibody, a recombinant vector, a recombinant strain and application, and relates to the technical field of biological medicines. The anti-PD-1 nano antibody and the recombinant nano antibody can specifically recognize and bind to a PD-1 antigen so as to block PD-1 / PD-L1 binding, have an in-vitro tumor cell killing effect, have killing strength related to action time, effect-target ratio and drug concentration, and also have an in-vivo anti-tumor effect.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to an anti-PD-1 nanobody, a coding gene, a recombinant nanobody, a recombinant carrier, a recombinant strain and applications. Background technique [0002] Programmed cell death protein-1 (programmed cell death protein 1, PD-1) is an important immunosuppressive molecule, belonging to the immunoglobulin superfamily CD28, encoded by human programmed cell death protein 1 gene, mainly expressed in activated T On the surface of cells, B cells and natural killer cells, interact with its ligand PD-L1 to down-regulate T cell activity and induce antigen tolerance to terminate or inhibit the immune response. Using PD-1 antibody can block PD-1 / PD- L1 signaling pathway, thereby releasing T cell inhibition and killing tumor cells. [0003] At present, specific monoclonal antibodies targeting PD-1 / PD-L1 are used to therapeutically block the binding of tumor cells to T lymphocyte ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C07K16/46C12N15/70C12N1/21G01N33/574G01N33/68A61K39/395A61P35/00B82Y5/00C12R1/19
CPCC07K16/2818C12N15/70G01N33/6854G01N33/574A61P35/00B82Y5/00C07K2319/30C07K2317/569C07K2317/76C07K2317/92G01N2333/70521A61K2039/505Y02A50/30
Inventor 徐广贤张爱君张艳婷蒋丹王丽燕李艳宁
Owner NINGXIA MEDICAL UNIV
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