Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

PD-1 (programmed death 1) nanoantibody and preparation method and application thereof

A nano-antibody, PD-1 technology, applied in the field of biomedicine, can solve the problems of long development cycle, easy degradation, and high storage cost of monoclonal antibodies, and achieve the effect of ensuring the original structure, activity and high activity

Active Publication Date: 2019-09-20
SHIHEZI UNIVERSITY
View PDF6 Cites 3 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • PD-1 (programmed death 1) nanoantibody and preparation method and application thereof
  • PD-1 (programmed death 1) nanoantibody and preparation method and application thereof
  • PD-1 (programmed death 1) nanoantibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 PD-1 nanobody, preparation method

[0056] S1: Synthesize and amplify PD-1 immune antigen sequence fragments.

[0057] First, obtain the PD-1 immune antigen sequence through the Uniprot database, see SEQ ID NO:1 for details. Then, use Primer Premier 5.0 software to design PD-1 upstream primers and PD-1 downstream primers, see the table below and SEQ ID NO:2 and SEQ ID NO:3 for details.

[0058]

[0059] 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.

[0060] The PCR system is: (primer concentration is 1OD dissolved in 400ul ddH 2 O)

[0061]

[0062]

[0063] PCR program:

[0064]

[0065] After the completion of PCR, 1% agarose gel electrophoresis was performed, and the fragments were recovered according to the operation of the recovery kit.

[0066] PCR results such as figure 2 As shown, the target ban...

Embodiment 2

[0183] The application of embodiment 2PD-1 nanobody

[0184] Cell experiment: Conduct preliminary in vitro cell pharmacodynamic evaluation of PD-1 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.

[0185] In the present embodiment, the cellular immunofluorescence method (semi-quantitative) is specifically adopted, and the specific scheme is as follows:

[0186] 1. When MC-38 cells grow confluent to 95%-100% on coverslips, take them out from the incubator.

[0187] 2. Wash 3 times with pre-warmed 1×PBS, 10 minutes each time.

[0188] 3. Fix with 4% formaldehyde at room temperature for 20-30 minutes.

[0189] 4. Wash 3 times with 1×PBS, 10 minutes each time.

[0190] 5. Permeabilize with 0.2% Triton X-100 for 2-5 minutes.

[0191] 6. Wash 3 times with 1×PBS, 10 minutes each time.

[0192] 7. Block with 5% BSA at room temperature for 30 minutes.

[0193] 8. Add ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention belongs to the technical field of biomedicine and particularly relates to a PD-1 (programmed death 1) nanoantibody and a preparation method and application thereof. The ability to bind with antigens can be improved or collaboratively enhanced through the PD-1 nanoantibody. The PD-1 nanoantibody has no complete antibody structure and lacks Fc end and Y-shaped structure; the antigens bound to the PD-1 nanoantibody are not easily recognizable and can escape easily from being caught by the immune system. The technical means includes mainly: performing antigen-vector connecting; transferring to competent cells, and extracting recombinant plasmids; transfecting to host bacteria for expressing; immunizing camels with antigen proteins; collecting peripheral blood, extracting total RNA of a lymphocyte sample, reversely 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 from the antibody immunization library; verifying the binding of the screened nanoantibodies with a PD-1 antigen.

Description

technical field [0001] The invention belongs to the field of biomedical technology, and in particular relates to PD-1 nanobody, preparation method and application thereof. Background technique [0002] PD-1 (programmed death 1) programmed death receptor 1 is an important immunosuppressive molecule. As a member of the immunoglobulin superfamily, it is a type I transmembrane glycoprotein with a molecular weight of 50-55kD. CD28 superfamily member. It was originally cloned from the apoptotic mouse T cell hybridoma 2B4.11. Immunomodulation targeting PD-1 is of great significance in anti-tumor, anti-infection, anti-autoimmune diseases and organ transplant survival. The human-derived PD-1 coding gene is located on chromosome 2, has 6 exons, and is expressed in T cells, B cells, monocytes, activated natural killer cells and other cells. It can inhibit the function of T cells through various ways. Immunomodulation targeting PD-1 is of great significance in anti-tumor, anti-infec...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00
CPCC07K16/2818A61P35/00C07K2317/569C07K2317/22C07K2317/92C07K2317/73
Inventor 陈创夫吴鹏
Owner SHIHEZI UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com