Single-domain antibody specifically for MMP9 protein ZnMc structural domain as well as product and application thereof

A single-domain antibody, MMP-9 technology, applied in the field of biomedicine, can solve the problems of incomplete specificity and efficacy, small modification space, and high immune heterogeneity

Active Publication Date: 2020-03-17
REGENECORE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The current MMP-9 antibodies are all traditional monoclonal antibodies. The traditional monoclonal antibody technology generally refers to the monoclonal antibody against the target protein prepared based on mouse hybridoma cells. The heavy chain and light chain or the Fab fragment of the antibody displayed at the same time replace the original hybridoma technology, but the final antibody structure is the same or similar to the traditional monoclonal antibody structure
The specificity and efficacy of traditional monoclonal antibodies are not completely satisfactory, in addition, the immune heterogeneity is high, and there is little room for transformation

Method used

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  • Single-domain antibody specifically for MMP9 protein ZnMc structural domain as well as product and application thereof
  • Single-domain antibody specifically for MMP9 protein ZnMc structural domain as well as product and application thereof
  • Single-domain antibody specifically for MMP9 protein ZnMc structural domain as well as product and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1: Construction of a single domain antibody library against MMP-9 protein:

[0091] (1) The protein ZnMc of 1mg MMP-9 zinc ion binding domain is mixed with Freund's complete adjuvant of equal volume (the purity detection result of the protein for immunization is as follows figure 1 shown), immunize a Bactrian camel in Alxa, Inner Mongolia, after the first immunization, use protein ZnMc mixed with an equal volume of Freund's incomplete adjuvant for immunization, immunize once a week, and then immunize 4 times consecutively, a total of 5 consecutive immunizations ; Then in the 6th and 7th times, animals were immunized with 1 mg MMP-9 full-length protein mixed with Freund's incomplete adjuvant in equal volumes. This immunization process was to stimulate the camels to produce antibodies against the ZnMc domain. This domain can activate the metalloprotease activity of MMP-9 after binding with Zn, and the protease activity can be blocked by obtaining an antibody that ...

Embodiment 2

[0094] Example 2: Screening against MMP-9 protein single domain antibody:

[0095] (1) Take 200 μL of recombinant TG1 cells to culture in 2×TY medium, add 40 μL of helper phage VCSM13 to infect TG1 cells during the period, and culture overnight to amplify the phages, use PEG / NaCl to precipitate the phages the next day, and centrifuge to collect the amplified phages ;

[0096] (2) NaHCO diluted in 100mM pH 8.3 3 500 μg of the ZnMc protein in the medium was coupled to the microtiter plate, placed overnight at 4°C, and a negative control well was set up at the same time;

[0097] (3) Add 200 μL of 3% skim milk the next day, and block at room temperature for 2 hours;

[0098] (4) After blocking, add 100 μl of amplified phage library (approximately 2×10 11 phage particles), at room temperature for 1 h;

[0099] (5) After acting for 1 hour, wash 5 times with PBS+0.05% Tween-20 to wash away unbound phage;

[0100] (6) Use trypsin at a final concentration of 25 mg / mL to dissociat...

Embodiment 3

[0101] Embodiment 3: use the enzyme-linked immunosorbent method (ELISA) of phage to screen the specific positive clone against MMP-9:

[0102] (1) According to the above-mentioned single-domain antibody screening method, three rounds of screening were performed on the MMP-9 protein. After the screening, the phage enrichment factor for the MMP-9 protein reached more than 10 (about 10,000), and the positive clones obtained from the screening Select 400 single colonies and inoculate them in 96-deep-well plates of TB medium containing 100 μg / mL ampicillin, and set up a blank control. After culturing to the logarithmic phase at 37°C, add IPTG with a final concentration of 1mM and culture at 28°C overnight;

[0103] (2) Use the osmotic swelling method to obtain the crude antibody; dilute the full-length MMP-9 protein and ZnMc protein to 100mM NaHCO at pH 8.3 3 Medium and coat 100 μg of protein in a microtiter plate overnight at 4°C;

[0104] (3) Transfer 100 μL of the antibody cru...

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Abstract

The invention, which relates to the technical field of biomedicine, particularly provides a single-domain antibody specifically for an MMP9 protein ZnMc structural domain as well as a product and application thereof. The single-domain antibody provided by the invention comprises a complementary determining region CDR including CDR1, CDR2 and CDR3; and the amino acid sequence of the CDR1 is any oneof SEQ ID NO.29 to SEQ ID NO.34; the amino acid sequence of the CDR2 is any one of SEQ ID NO. 35 to SEQ ID NO. 41; and the amino acid sequence of the CDR3 is any one of SEQ ID NO. 42 to SEQ ID NO. 50. The affinity of the single-domain antibody is obvious and can replace the traditional monoclonal antibody for rapid detection to detect MMP-9 protein or changing the activity of the MMP-9 protein.

Description

technical field [0001] The present invention relates to the field of biomedical technology, in particular to a single-domain antibody specific for the ZnMc domain of MMP9 protein, as well as its products and applications. Background technique [0002] Matrix metalloproteinases (MMPs) belong to a family of extracellular enzymes involved in the formation and remodeling of the extracellular matrix. These enzymes contain a conserved catalytic domain in which the zinc atom is coordinated via three histidine residues. Members of this family are organized into groups including collagenases, gelatinases, stromelysins, stromelysins, amelysins, and membrane MMPs. [0003] MMP-9 (matrix metalloproteinase-9) belongs to the gelatinase group of matrix metalloproteinases. The MMP-9 gene is located on chromosome 20q11.1-13.1, 26-27kbp, with 13 exons and 9 introns. The main function of MMP-9 is to degrade and remodel the dynamic balance of the extracellular matrix (extracellular matrix). ...

Claims

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Application Information

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IPC IPC(8): C07K16/40C12N15/13A61K39/395A61P35/00A61P29/00A61P37/02
CPCA61P29/00A61P35/00A61P37/02C07K16/40C07K2317/24C07K2317/565C07K2317/567C07K2317/569C07K2317/76C07K2317/92
Inventor 苏志鹏孟巾果赵泽英
Owner REGENECORE BIOTECH CO LTD
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