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Nano-antibody B3 of anti-human transforming growth factor beta1, as well as preparation method and application thereof

A transforming growth factor, nanobody technology, applied in microorganism-based methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problems of high immunogenicity, poor solubility, easy to be degraded by protease, etc. The effect of strong sex and high affinity

Active Publication Date: 2020-07-24
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • 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

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  • Nano-antibody B3 of anti-human transforming growth factor beta1, as well as preparation method and application thereof
  • Nano-antibody B3 of anti-human transforming growth factor beta1, as well as preparation method and application thereof
  • Nano-antibody B3 of anti-human transforming growth factor beta1, as well as preparation method and application thereof

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 human TGF-β1 standard full-length DNA sequence used in this example (including N-terminal signal peptides (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 1173bp, as shown in SEQ ID NO: 1 in the sequence listing) stored in the Gateway Cloning vector (obtained by conventional Gateway gene cloning technology).

[0046] This embodiment specifically comprises the following steps:

[0047] 1.1, first utilize the Snapgene program to design the upstream primer F and downstream primer R that are used to amplify and obtain the standard full-length DNA sequence of TGF-β1, and introduce Age I and EcoR I restriction sites respectively (the following upstream primer F and downstream primer The underlined part in the R sequence) and its protective b...

Embodiment 2

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

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

[0059] 2.1. For the first immunization, 1 mg of TGF-β1 purified protein (obtained in Example 1) was mixed with complete Freund’s adjuvant in an equal volume of 1:1 and fully emulsified, and the alpaca was immunized by subcutaneous injection; The β1 purified protein was mixed with incomplete Freund's adjuvant in an equal volume of 1:1 for 4 booster immunizations; a total of 5 immunizations were performed, with a single interval of 20 days. A small amount of alpaca peripheral blood was extracted for titer detection. When the immune titer was greater than 1:60000, booster immunization was carried out again. After 24 hours, 100 mL of anticoagulant blood was collected from the jugular vein of the immunized alpaca. ...

Embodiment 3

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

[0067] 3.1: Simplified steps of affinity panning:

[0068] (1) Coat the immunotube 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% skimmed milk added to PBS), incubate at 37°C for 2 hours, pour off the blocking solution, wash the tube 3 times with PBS, and pat dry.

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

[0072] (5) Discard the phage library in the tube, wash 3 times with PBST, then wash 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 panning ste...

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Abstract

The invention discloses a nano-antibody B3 of anti-human transforming growth factor beta1, as well as a preparation method and application thereof, and belongs to the field of biological pharmacy. According to the preparation method, the nano-antibody of anti-human transforming growth factor beta1 with relatively high binding force with antigen is filtered out by adopting the phage display technology and the biological panning technology. The nano-antibody can specifically bind with three modes of natural TGF-beta1 protein, namely LAP precursor, TGF-beta monomer and dipolymer, has the characteristics of strong specificity and high affinity, can effectively neutralize autocrine or paracrine TGF-beta1 in a tumor microenvironment, and provides a gene tool to tumor immunotherapy, wound healingpromotion, tissue repair and embryonic development promotion and other aspects.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to an anti-human transforming growth factor β1 nanobody and its preparation method application, in particular to an anti-human transforming growth factor β1 with high specificity recognition and high affinity for human transforming growth factor β1 Nanobody and its preparation method and application. Background technique [0002] Transforming growth factor β (TGF-β) is one of the cytokines that regulate 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 the functions of regulating cell proliferation and differentiation, and controlling the synthesis and degradation of extracellular matrix proteins. It also participates in embryonic and skeletal development, tissue remodeling, and wound healing. However, during the malignant progression of tumors, the highly activate...

Claims

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Application Information

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Patent Type & Authority Applications(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