Method for preparing PD-L1 (programmed death-ligand 1) nanoantibody by immunizing alpacas with PD-L1 antigen

A PD-L1 and nanobody technology, applied in the field of antibodies, can solve the problems of high storage cost, instability, and non-repeatable application

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

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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
[0008] (2) The monoclonal antibody is relatively large in size and cannot enter the tumor tissue well
[0009] (3) The monoclonal antibody development cycle is long, the production cost is high, and the output is low
[0010] (4) Monoclonal antibody drugs are expensive, complex in research and development, complex in humanizatio...

Method used

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  • Method for preparing PD-L1 (programmed death-ligand 1) nanoantibody by immunizing alpacas with PD-L1 antigen
  • Method for preparing PD-L1 (programmed death-ligand 1) nanoantibody by immunizing alpacas with PD-L1 antigen
  • Method for preparing PD-L1 (programmed death-ligand 1) nanoantibody by immunizing alpacas with PD-L1 antigen

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Embodiment 1

[0158] Example 1. The carrier construction provided by the embodiment of the present invention is specifically:

[0159] 1. Target fragment amplification is specifically:

[0160] (1) 34 primers were designed and synthesized, and a sufficient amount of the target product was amplified by PCR.

[0161] (2) The PCR reaction uses pfu high-temperature polymerase.

[0162] The amount of each component of PCR: (primer concentration is 1OD dissolved in 400ul ddH2O).

[0163] Reaction system: 50ul. Primer mix, (1 / 34) 0.4ul x 13.6 total 8ul. 10X pfu Buffer, 5ul. Each upstream and downstream primers, each 2ul. Pfu, 0.4ul (5u / ul). ddH2O, add water to 50ul respectively.

[0164] The method amplification specific steps of target fragment PCR are:

[0165] (1) The first round of PCR procedure:

[0166]

[0167] 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.

[0168] (2) The second round of...

Embodiment 2

[0187] The construction of the nanobody library provided by the embodiments of the present invention is specifically:

[0188] 1. Experimental design

[0189] 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

[0211] The protein identification and expression provided by the embodiments of the present invention are specifically:

[0212] (1) Construction of mammalian cell expression vector

[0213] 1. Amplify and extract the vector plasmid containing the target gene.

[0214] 2. Subcloning into eukaryotic expression vector pcDNA3.1.

[0215] 3. Sequencing to verify the accuracy of the constructed plasmid.

[0216] 4. Obtain the recombinant plasmid pcDNA3.1 by pumping.

[0217] (2) Mammalian cell culture, protein expression and purification experiments

[0218] 1. Cell lines and materials

[0219] Cell line: HEK293 cells.

[0220] Medium: DMEM (10% serum), DMEM (serum-free).

[0221] Petri dish: 10cm dish or 15cm dish

[0222] 2. HEK293 cell transfection (10cm dish)

[0223] (1) 24 hours before transfection, the total amount of cells in a 4-5X106 / 10cm culture dish is plated, and the transfection can be carried out when the cells are growing well and the density of adherent cells...

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Abstract

The invention belongs to the technical field of antibodies and discloses a method for preparing PD-L1 (programmed death-ligand 1) nanoantibody by immunizing alpacas with a PD-L1 antigen. The method comprises: designing two synthetic primers, amplifying by PCR (polymerase chain reaction) to obtain sufficient target products for enzyme digestion, linking target fragments to a vector, converting, and carrying out screening and cloning; carrying out protein verifying and expressing; constructing an antibody library; displaying a VHH antibody library through an M13 bacteriophage display system which is composed of a pMECS phagemid vector, E. coli TG1 and an M13KO7 helper phage. In the phasmid vector pMECS, a sequence before a Pst I enzyme digestion site is a coding sequence for part of amino acids in first frame regions of pelB secretory signal peptides and antibodies; the pelB signal peptides can guide subsequent polypeptides secreted to periplasmic cavities; a sequence after a Not I enzyme digestion site is a coding sequence for HA and 6*His tags; the method is suitable for purification or detection of fusion proteins.

Description

technical field [0001] The invention belongs to the technical field of antibodies, and in particular relates to a method for preparing PD-L1 nanobody by immunizing alpaca with PD-L1 antigen. Specifically, it relates to a method for preparing PD-L1 nanobody by immunizing alpaca with mammalian cells expressing PD-L1 antigen. Background technique [0002] At present, the existing technologies commonly used in the industry are as follows: [0003] At present, some anti-PD-L1 monoclonal antibodies have been approved for the treatment of cancer. For example, the PD-L1 inhibitor Tecentriq (atezolizumab or MDL3280a), the approved indication is bladder cancer (urothelial carcinoma). The expression of PD-L1 is increased in most tumor tissues, such as breast cancer, non-small cell lung cancer, and melanoma. The expression of PD-L1 in tumor cells is also considered to be a prognostic factor for many types of malignant tumors. In order to block the combination of PD-L1 and receptor PD...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N15/85A61K47/68
CPCC07K16/2827C12N15/85A61K47/6849C07K2317/22C07K2317/569C12N2800/107C12N15/1037C07K2317/70C12Q1/686
Inventor 陈创夫吴鹏
Owner SHIHEZI UNIVERSITY
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