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Single-domain antibodies, products and applications specific to the zinc ion-binding domain of mmp-9 protein

An MMP-9, single-domain antibody technology, applied in the biomedical field, can solve the problems of small transformation space, high immune heterogeneity, incomplete specificity and efficacy, etc., and achieve simple transformation and immune heterogeneity. The effect of low, good binding activity

Active Publication Date: 2021-07-02
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 antibodies, products and applications specific to the zinc ion-binding domain of mmp-9 protein
  • Single-domain antibodies, products and applications specific to the zinc ion-binding domain of mmp-9 protein
  • Single-domain antibodies, products and applications specific to the zinc ion-binding domain of mmp-9 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1: Construction of a single domain antibody library against MMP-9 protein:

[0090] (1) The protein of 1 mg MMP-9 zinc ion binding domain is mixed with an equal volume of Freund's complete adjuvant (the purity test results of the protein used for immunization are as follows: figure 1 shown), to immunize a Bactrian camel in Alxa, Inner Mongolia. After the first immunization, the protein was mixed with an equal volume of Freund's incomplete adjuvant for immunization, once a week, and then 4 consecutive immunizations, for 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 zinc ion binding domain , this domain can be combined with Zn to activate the metalloprotease activity of MMP-9, and the antibody that can block the combination of MMP-9 and Z...

Embodiment 2

[0093] Example 2: Screening against MMP-9 protein single domain antibody:

[0094] (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 ;

[0095] (2) NaHCO diluted in 100mM pH 8.3 3 500 μg of the MMP-9 zinc ion binding domain protein was coupled to the microtiter plate, placed overnight at 4°C, and a negative control well was set up at the same time;

[0096] (3) Add 200 μL of 3% skim milk the next day, and block at room temperature for 2 hours;

[0097] (4) After blocking, add 100 μl of amplified phage library (approximately 2×10 11 phage particles), at room temperature for 1 h;

[0098] (5) After acting for 1 hour, wash 5 times with PBS+0.05% Tween-20 to wash away unbound phage;

[0099] (6) Use trypsin at a final concentration of 25 mg / mL t...

Embodiment 3

[0100] Embodiment 3: use the enzyme-linked immunosorbent method (ELISA) of phage to screen the specific positive clone against MMP-9:

[0101] (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;

[0102] (2) Use the osmotic swelling method to obtain the crude antibody; dilute the MMP-9 full-length protein and the MMP-9 zinc ion binding domain protein to 100mM NaHCO at pH 8.3 3 Medium and coat 100μg of protein in a microtiter plate overnight at 4°C;

[0103] (3) Transfe...

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Abstract

The invention relates to the field of biomedical technology, specifically, it provides a single-domain antibody specific for the zinc ion binding domain of MMP-9 protein, as well as its products and applications. The single domain antibody provided by the present invention includes complementarity determining region CDR including CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is as any one of SEQ ID NO.29-SEQ ID NO.34; the amino acid sequence of CDR2 is as SEQ ID NO. Any one of 35-SEQ ID NO.41; the amino acid sequence of CDR3 is any one of SEQ ID NO.42-SEQ ID NO.50. The single-domain antibody has obvious affinity and can replace the traditional monoclonal antibody for rapid expression to detect MMP‑9 protein or change its activity.

Description

technical field [0001] The invention relates to the field of biomedical technology, in particular to a single-domain antibody specific for the zinc ion binding domain of MMP-9 protein, as well as its product and application. 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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Patent Type & Authority Patents(China)
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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