Anti-PD-L1 nano antibody and coding sequence, preparation method and application thereof

A technology of PD-L1 and nano-antibody, which is applied in the field of biomedicine, can solve the problems of large molecular weight, difficulty penetrating tissues, and long development cycle of monoclonal antibodies, and achieve the effect of high specificity and high detection sensitivity

Active Publication Date: 2019-01-25
JIANGSU TARGET BIOMEDICINE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, monoclonal antibodies have disadvantages such as long development cycle, high production cost, large molecular weight, and difficulty in penetrating tissues.

Method used

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  • Anti-PD-L1 nano antibody and coding sequence, preparation method and application thereof
  • Anti-PD-L1 nano antibody and coding sequence, preparation method and application thereof
  • Anti-PD-L1 nano antibody and coding sequence, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1 Anti-PD-L1 nanobody library construction

[0032] (1) Mix 1 mg of the His-tagged PD-L1 extracellular segment (19-239) antigen with an equal volume of Freund's adjuvant, and immunize a Xinjiang Bactrian camel once a week for a total of 5 times to stimulate B Cells expressing antigen-specific nanobodies;

[0033] (2) After the immunization, extract 100mL camel peripheral blood lymphocytes and extract total RNA;

[0034] (3) Reverse transcription PCR

[0035] I. adopt RT-PCR kit, reverse transcribe the total RNA of peripheral blood lymphocytes into cDNA,

[0036] II. The cDNA was amplified by nest-PCR to obtain the VHH fragment of camelid heavy chain antibody.

[0037] Using the cDNA obtained above as a template and nest-PCR1up (SEQ ID NO: 28) and nest-PCR1down (SEQ ID NO: 29) as primers, the first round of PCR amplification was performed. After the PCR reaction was completed, the PCR product was detected by agarose gel electrophoresis. The results of gel electrophor...

Embodiment 2

[0099] 1 Vector construction, expression and purification of anti-PD-L1 nanobody protein, anti-PD-L1 nanobody and wasabi fusion protein

[0100] (1) Vector construction, expression and purification of anti-PD-L1-VHH nanobodies (anti-PD-L1Nb15, anti-PD-L1Nb22, anti-PD-L1Nb28).

[0101] The nucleotide sequence of the anti-PD-L1 nanobody was digested with EcoRI and HindⅢ, the digested product was recovered by agarose gel electrophoresis, and inserted into PET32a to construct the expression vector of the anti-PD-L1 nanobody ; Transformed into BL21(DE3), IPTG induced expression. Centrifuge the bacterial liquid to obtain bacterial pellet, resuspend in lysis buffer, ultrasonically disrupt, and collect the supernatant by centrifugation. Purified anti-PD-L1 nanobody was obtained by Ni-IDA affinity chromatography. Then use SDS-PAGE electrophoresis detection (see figure 2 ).

[0102] (2) Construct the fusion protein expression vector of anti-PD-L1Nb28 and wasabi, and prepare wasabi-...

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Abstract

The invention discloses an anti-PD-L1 nano antibody and a coding sequence, a preparation method and application thereof. An amino acid sequence of an antibody VHH chain is as shown in SEQ ID NO:2, SEQID NO:11 or SEQ ID NO:20. Compared with the prior art, the anti-PD-L1 nano antibody has the following advantages: (1) the anti-PD-L1 nano antibody has high specificity, and high detection sensitivity; and (2) the antibody can be efficiently expressed in escherichia coli, so a foundation is provided for follow-up mass production of an immunoreagent and a tumor drug composition.

Description

technical field [0001] The invention belongs to the field of biomedical technology, and relates to a novel PD-L1 antibody or a functional fragment thereof, specifically anti-PD-L1 nanobody and its coding sequence, preparation method and application. Background technique [0002] Programmed death 1 ligand 1 (programmed death 1 ligand 1, PD-L1), also known as CD274, B7-H1, is a member of the B7 family, a ligand of PD-1, and a type I transmembrane protein. Under normal circumstances, PD-1 on the surface of T cells can inhibit the function of T lymphocytes, thereby inhibiting autoimmune responses and preventing the occurrence of autoimmune diseases. However, in tumors, after PD-L1 expressed by tumor cells binds to PD-1 on the surface of T cells, it can inhibit T lymphocytes, lead to down-regulation of T cell proliferation, and even induce T cell apoptosis to promote tumor immunity. escape. Blocking the negative regulatory pathway of PD-1 / PD-L1 can activate the function of the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13G01N33/68A61K39/395A61P35/00
CPCA61K39/00A61P35/00C07K16/2827C07K2317/22C07K2317/33C07K2317/569G01N33/6872G01N2333/70532
Inventor 李淑锋姜昆鹏王婷
Owner JIANGSU TARGET BIOMEDICINE RES INST
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