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A CD105 nanobody nb59

A nanobody and DNA molecule technology, applied in the field of biomedicine or biopharmaceuticals, can solve the problem of lack of nanobody and achieve the effect of good specificity and high affinity

Inactive Publication Date: 2020-10-13
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 CD105 nanobody, DNA molecule encoding the CD105 nanobody and the use of the nanobody, etc.

Method used

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  • A CD105 nanobody nb59
  • A CD105 nanobody nb59
  • A CD105 nanobody nb59

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Construction of a Nanobody library for CD105:

[0027] (1) First synthesize CD105 polypeptide, mix 1 mg CD105 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 CD105 nanobody library was constructed and the storage capacity was determined, and the storage capacity was 1.85×10 8 .

Embodiment 2

[0031] Example 2: Nanobody screening process against CD105:

[0032] (1) Dissolve in 100mM NaHCO 3 , 20ug CD105 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 CD105, and infect Escherichia coli TG1 in logarithmic phase growth, culture at 37°C for 1 hour, 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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Abstract

The invention discloses a CD105 nano antibody aiming at CD105 polypeptide molecular epitope, further discloses a coding gene sequence of the CD105 nano antibody, a CD105 nano antibody expression carrier and a host cell and also discloses application of the CD105 nano antibody. By the CD105 nano antibody, the gene sequence, the host cell and the like, efficient expression of the CD105 nano antibody in escherichia coli can be realized, high specificity in immunoreactions with CD105 and high affinity can be realized, and application to preparation of CD105 detection agents, antitumor medicines or the like is realized.

Description

technical field [0001] The invention belongs to the technical field of biomedicine or biopharmaceuticals, and more specifically relates to a nanobody (Nb59) directed at the epitope molecule of CD105 polypeptide molecule, its coding sequence and application. Background technique [0002] CD105, also known as endoglin, is a glycoprotein expressed on the cell membrane of endothelial cells. It is one of the components of the transforming growth factor β (transform growth factor, TGF-β) receptor complex, but it can exist independently on the cell surface. CD105 contains 633 amino acids with a relative molecular mass of about 68051. It is a protein related to proliferation and can be induced by hypoxia. Its outer part of the cell membrane contains 561 amino acids, and the transmembrane region contains 25 amino acids. The portion contains 47 amino acids and constitutes the tail of CD105. In the extracellular part of CD105, there are four potential glycosylation sites, which are am...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13G01N33/68G01N33/574A61K39/395A61P35/00B01J20/24B01D15/08B82Y30/00
CPCB01D15/08B01J20/24B82Y30/00C07K16/2863C07K2317/565C07K2317/567C07K2317/569
Inventor 赵永祥卢小玲
Owner GUANGXI MEDICAL UNIVERSITY
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