Supercharge Your Innovation With Domain-Expert AI Agents!

Anti-human transforming growth factor beta 1 nanobody b3 and its preparation method and application

A transforming growth factor and nanobody technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problems of poor solubility, high immunogenicity, reduction of tumor matrix protein synthesis and fibroblast The quantity and other issues, to achieve the effect of high affinity and strong specificity

Active Publication Date: 2022-07-15
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Studies have shown that blocking antibodies targeting TGF-β1 signaling pathway, such as anti-TGF-β1 antibody Fresolimumab (GC1008), anti-TGF-β1 receptor antibody Lapatimab (AB0213), etc., can significantly reduce the synthesis and formation of tumor matrix proteins. However, most of the existing complete antibodies have the disadvantages of high immunogenicity, poor solubility, easy adhesion and aggregation, and easy degradation by proteases, which limit the ability of these protein antibodies to block TGF. Application of -β1 in turn inhibiting tumor growth and migration

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
  • Anti-human transforming growth factor beta 1 nanobody b3 and its preparation method and application
  • Anti-human transforming growth factor beta 1 nanobody b3 and its preparation method and application
  • Anti-human transforming growth factor beta 1 nanobody b3 and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1. Construction of eukaryotic expression plasmid cFUGW-TGF-β1 and protein expression and purification

[0045] The standard full-length DNA sequence of human TGF-β1 used in this example (including N-terminal signal peptide (signalpeptides, 1-39aa), LAP precursor structure (30-278aa) and C-terminal TGF-β monomer (279-390aa) ) three parts, the length of the coding nucleotide sequence is 1173 bp, as shown in SEQ ID NO: 1 in the sequence listing) and stored in the Gateway Cloning vector (obtained by conventional Gateway gene cloning technology).

[0046] This embodiment specifically includes the following steps:

[0047] 1.1. First use Snapgene program to design upstream primer F and downstream primer R for amplifying the standard full-length DNA sequence of TGF-β1, and introduce Age I and EcoR I restriction sites (the following upstream primer F and downstream primer respectively) The underlined part in the R sequence) and its protective base; in addition, the Koz...

Embodiment 2

[0057] Example 2: Construction of alpaca phage VHH display library

[0058] This example uses the purified protein of TGF-β1 obtained in the above Example 1 to immunize alpacas to construct a VHH gene library, which specifically includes the following steps:

[0059] 2.1. Primary immunization 1 mg of TGF-β1 purified protein (obtained in Example 1) was mixed with complete Freund's adjuvant 1:1 in equal volume and fully emulsified, and the alpacas were immunized by subcutaneous injection; β1 purified protein was mixed with an equal volume of incomplete Freund's adjuvant for 4 booster immunizations; a total of 5 times of immunization, with a single interval of 20 days. A small amount of alpaca peripheral blood was drawn for titer detection. When the immune titer was greater than 1:60,000, booster immunization was performed again. After 24 hours, 100 mL of anticoagulation was collected from the jugular vein of the immunized alpaca. Peripheral blood lymphocytes were extracted from...

Embodiment 3

[0066] Example 3: Screening of positive clones expressing Nanobody TGF-β1-VHH

[0067] 3.1: Simplified steps for affinity panning:

[0068] (1) Coat the immune tube with the purified TGF-β1 protein obtained in Example 1, overnight at 4°C.

[0069] (2) Wash the tube 3 times with PBS and pat dry.

[0070] (3) Block with 3% MPBS (3% skim milk added to PBS), incubate at 37° C. for 2 h, pour off the blocking solution, wash the tube three times with PBS, and pat dry.

[0071] (4) Add the alpaca phage VHH display library (phage library) prepared in Example 2 to the sealed immune tube, 2 ml / tube, shake gently for 30 min, and then incubate for 1.5 h.

[0072] (5) Discard the phage library in the tube, wash 3 times with PBST, then 3 times with PBS, and pat dry.

[0073] (6) Add host strain TG1 to elute the bound phage library. This completes the first round of panning and obtains the primary screening antibody library.

[0074] (7) Repeat the above-mentioned panning steps 4), 5) an...

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

No PUM Login to View More

Abstract

The invention discloses a nanobody B3 against human transforming growth factor beta 1 and a preparation method and application thereof, belonging to the field of biopharmaceuticals. The provided preparation method adopts the phage display technology and the biopanning technology to screen out the anti-human transforming growth factor beta 1 nanobody with high binding force to the antigen. The nanobody can specifically bind to three forms of natural TGF-β1 protein: LAP precursor, TGF-β1 monomer and dimer, and has the characteristics of strong specificity and high affinity, which can effectively neutralize tumors Autocrine or paracrine TGF-β1 in the microenvironment provides a genetic tool for tumor immunotherapy, promoting wound healing, and promoting tissue repair and embryonic development.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, relates to an anti-human transforming growth factor beta 1 nanobody and a preparation method and application thereof, and particularly relates to an anti-human transforming growth factor beta 1 with high specific recognition and high affinity for human transforming growth factor beta 1 Nanobodies and their preparation methods and applications. Background technique [0002] Transforming growth factor beta (TGF-beta) is one of the cytokines that regulates cell growth, differentiation and immune function. At present, the human TGF-β superfamily has four members: TGF-β1, TGF-β2, TGF-β3, and TGF-β1β2. Among them, TGF-β1 has functions such as regulating cell proliferation and differentiation, and controlling the synthesis and degradation of extracellular matrix proteins. It is also involved in embryogenesis and bone formation, tissue remodeling and wound healing. However, during the malignant progre...

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 Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/70C12N1/21A61K39/395A61P35/00C12R1/19
CPCC07K16/2863C12N15/70A61P35/00C07K2317/569C07K2317/92A61K2039/505
Inventor 张建民王玥陈慧胡瑜许依何维
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

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

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com