A FAP nanobody nb26

A technology of nanobodies and DNA molecules, applied in the field of biomedicine or biopharmaceuticals, to achieve high affinity and good specificity

Active Publication Date: 2020-09-25
GUANGXI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a FAP nanobody, and at the same time provide a DNA molecule encoding the FAP nanobody and the use of the nanobody for the lack of nanobodies against the FAP epitope in the prior art.

Method used

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  • A FAP nanobody nb26
  • A FAP nanobody nb26
  • A FAP nanobody nb26

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Construction of a Nanobody library for FAP:

[0027] (1) First synthesize FAP polypeptide, mix 1mg FAP with Freund's adjuvant in equal volume, immunize a Xinjiang dromedary, once a week, immunize 7 times in total, and stimulate B cells to express antigen-specific nanobodies;

[0028] (2) After the 7 times of immunization, extract 100mL camel peripheral blood lymphocytes and extract total RNA;

[0029] (3) Synthesize cDNA and amplify VHH by nested PCR;

[0030] (4) Use restriction enzymes PstI and NotI to digest 20ug pComb3 phage display vector (supplied by Biovector China Plasmid Vector Strain Cell Gene Collection Center) and 10ug VHH and connect the two fragments; (5) Transform the ligated product into electroporation In state cell TG1, the FAP nanobody library was constructed and the storage capacity was determined, and the storage capacity was 1.85×10 8 .

Embodiment 2

[0031] Embodiment 2: Nanobody screening process against FAP:

[0032] (1) Dissolve in 100mM NaHCO 3 , 20ug FAP in pH 8.2 was coupled to a NUNC microtiter plate, and placed overnight at 4°C;

[0033] (2) Add 100uL 0.1% casein the next day, and block at room temperature for 1-3h;

[0034](3) After 1-3h, add 100uL phage (5×10 11 tfu immunized camel nanobody phage display gene library), at room temperature for 1-2h;

[0035] (4) Wash 4-6 times with 0.05% PBS+Tween-20 to wash off unbound phages;

[0036] (5) Use 100mM TEA (triethylamine) to dissociate the phage that specifically binds to FAP, and infect Escherichia coli TG1 in logarithmic phase growth, culture at 37°C for 1h, produce and purify the phage for the next round For screening, the same screening process was repeated for 3-5 rounds to gradually obtain enrichment.

Embodiment 3

[0037] Embodiment 3: use the enzyme-linked immunosorbent method (ELISA) of phage to screen specificity single positive clone:

[0038] (1) From the cell culture dish containing phage after the above 3-5 rounds of selection, pick 96 single colonies and inoculate them in TB medium containing 100 micrograms per milliliter of ampicillin (1 liter of TB medium contains 2.3 grams of phosphoric acid Potassium dihydrogen, 12.52 grams of dipotassium hydrogen phosphate, 12 grams of peptone, 24 grams of yeast extract, 4 milliliters of glycerol), after growing to the logarithmic phase, add isopropylthiogalactoside ( IPTG), cultivate overnight at 30-35°C.

[0039] (2) Use the infiltration method to obtain the crudely extracted antibody, and transfer the antibody to an antigen-coated ELISA plate, and place it at room temperature for 1-1.5 hours.

[0040] (3) Wash off unbound antibodies with PBST, add primary mouse anti-HA tagantibody (purchased from Beijing Kangwei Century Biotechnology Co....

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PUM

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Abstract

The invention discloses a FAP (familial adenomatous polyposis) nanometer antibody specific to FAP polypeptide epitope, while also discloses a gene sequence for encoding the FAP nanometer antibody and an expression carrier for expressing the nanometer antibody, and a host cell, and further discloses an application of the FAP nanometer antibody. Through the FAP nanometer antibody, the gene sequence and the host cell, and others disclosed in the invention, the FAP nanometer antibody can be expressed efficiently in escherichia coli, and have good specificity with FAP immunoreaction and high appetency; the FAP nanometer antibody can be applied to prepare FAP detection reagent or anti-cancer drug, and others.

Description

technical field [0001] The invention belongs to the technical field of biomedicine or biopharmaceuticals, and more specifically relates to a nanobody (Nb26) directed at the epitope molecule of the FAP polypeptide molecule, its coding sequence and application. Background technique [0002] Fibroblast activation protein (fibroblast activation protein, FAP) is a surface antigen specifically expressed by tumor-associated fibroblasts. It has dipeptidyl peptidase and collagenase activities. Immunosuppressive effect. FAP is composed of 761 amino acids, and the glycosylation level is different in different mammalian cells, and the relative molecular mass of the monomer is 8.8×10 4 , 9.5×10 4 , 9.7×10 4 and other reports, the relative molecular mass of the dimer is 1.7×10 5 . The structure of FAP is divided into three parts: the cytoplasmic region, the transmembrane region and the extracellular region. The cytoplasmic region is a short peptide chain composed of the first 6 amino...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/40C12N15/13G01N33/574G01N33/573A61K39/395A61P35/00B01J20/24B01D15/08B82Y30/00
CPCB01D15/08B01J20/24B82Y30/00C07K16/40C07K2317/565C07K2317/567C07K2317/569
Inventor 卢小玲赵永祥
Owner GUANGXI MEDICAL UNIVERSITY
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