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Nanobody pd-1/nb52 against pd-1 and its preparation method and application

A technology of nano-antibodies and recombinant vectors, which is applied in botany equipment and methods, biochemical equipment and methods, antibodies, etc., and can solve problems such as high production costs, poor stability and easy degradation, and long research and development cycles.

Active Publication Date: 2019-10-11
GUANGXI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, there are many problems in the application of antibody drugs, such as long research and development cycle, high production cost, difficulty in large-scale production, poor stability and easy degradation, high storage cost, easy contamination, high maintenance cost, and immunogenicity, etc. its scope of clinical application

Method used

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  • Nanobody pd-1/nb52 against pd-1 and its preparation method and application
  • Nanobody pd-1/nb52 against pd-1 and its preparation method and application
  • Nanobody pd-1/nb52 against pd-1 and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0074] Embodiment 1, the preparation of nanobody

[0075] The present invention provides a nanobody derived from camel, its name is PD-1 / Nb52, the amino acid sequence of the nanobody PD-1 / Nb52 is shown in SEQ ID No.8 in the sequence listing, represented by SEQ ID No. 9 nucleotide sequence encoding.

[0076] Nucleotide electrophoresis of Nanobody PD-1 / Nb52 as shown in figure 1 As shown, the first lane is a molecular marker of 2000bp, the rest of the lanes are PCR products, and the PCR product band is about 400bp.

[0077] Replace the DNA fragment between the PstI and NotI recognition sequences of the vector pComb3 (Biovector product) with the DNA molecule shown in SEQID No.9, and keep the other sequences unchanged to obtain the recombinant vector pComb3-PD-1 / Nb52, pComb3-PD- The only difference between 1 / Nb52 and pComb3 is that the DNA segment between the PstI and NotI recognition sequences of pComb3 is replaced by the DNA molecule shown in SEQ ID No.9. The recombinant vect...

Embodiment 2

[0085] Example 2, Determination of Nanobody and PD-1 Binding Rate

[0086] Determination of the binding rate of Nanobody PD-1 / Nb52 to PD-1 (direct method)

[0087] 293T cells stably transfected with PD-1 were used to detect the binding rate of Nanobody PD-1 / Nb52 to PD-1, and the Nanobody PD-1 / Nb52 (1 μg) in Example 1 was added to 1-6×10 6 The above-mentioned 293T cells were incubated at 4°C in the dark for 20-40min, washed twice with PBS, added 5 μl PE anti-HA tag antibody (abcam, Clone: ​​20B12) and incubated at 4°C for 20-40min, washed twice with PBS, and the samples were On the BACKMAN flow cytometer, the results are as follows Figure 3B As shown, 293T cells not transfected with PD-1 were used as a control such as Figure 3A shown. Figure 3A is the binding percentage of blank control and PD-1 Nanobody PD-1 / Nb52 to 293T cells not transfected with PD-1; Figure 3B is the binding percentage of blank control and PD-1 nanobody PD-1 / Nb52 respectively stably transfecting PD-...

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Abstract

Disclosed are an anti-PD-1 nanobody PD-1 / Nb52, a preparation method therefor and a use thereof. The nanobody PD-1 / Nb52 can be highly expressed in Escherichia coli, and can be used for preparing tumor inhibitors or tumor cell inhibitors, or drugs for inhibiting PD-1 activity or promoting T cell proliferation.

Description

technical field [0001] The invention relates to anti-PD-1 nanobody PD-1 / Nb52 in the field of biomedicine and its preparation method and application. Background technique [0002] Programmed death receptor 1 (programmed death 1, PD-1) is a transmembrane glycoprotein molecule expressed on the surface of activated T cells, which is a member of the CD28 superfamily and negatively regulates the proliferation of T cells. play an important regulatory role. PD-1 is mainly expressed in activated T cells and B cells, and is a surface receptor of activated T cells. PD-1 has two ligands, programmed cell death-ligand 1 (PD-L1, Also known as B7-H1) and programmed cell death-ligand 1 (PD-L2, also known as B7-DC). The tumor microenvironment in the body will induce infiltrating T cells to highly express PD-1 molecules, and tumor cells will highly express PD-1 ligands PD-L1 and PD-L2, resulting in the continuous activation of PD-1 pathway in the tumor microenvironment. After -L1 is combine...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
Inventor 卢小玲杨晓梅段斯亮
Owner GUANGXI MEDICAL UNIVERSITY