CD105 nano antibody Nb68

A technology of nanobodies and DNA molecules, applied in the field of biomedicine or biopharmaceuticals, can solve the problems of lack of nanobodies and achieve the effect of good specificity and high affinity

Inactive Publication Date: 2017-07-07
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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  • CD105 nano antibody Nb68
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  • CD105 nano antibody Nb68

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Construction of Nanobody library targeting CD105:

[0027] (1) First, synthesize CD105 polypeptide, mix 1 mg CD105 and Freund's adjuvant in equal volumes, immunize a Xinjiang dromedary camel, once a week, immunize 7 times to stimulate B cells to express antigen-specific Nanobodies;

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

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

[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) and 10ug VHH and connect the two fragments; (5) Transform the ligation product into electrotransduction In the cell TG1, construct a CD105 nanoantibody library and determine its storage capacity. The storage capacity is 1.85×10 8 .

Embodiment 2

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

[0032] (1) Will be dissolved in 100mM NaHCO 3 , 20ug of CD105 in pH 8.2 is coupled to the NUNC plate, placed overnight at 4°C;

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

[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) Dissociate the phage that specifically binds to CD105 with 100mM TEA (triethylamine), and infect E. coli TG1 growing in the logarithmic phase, incubate at 37℃ for 1h, produce and purify the phage for the next round Screening, the same screening process is repeated 3-5 rounds, and enrichment is gradually obtained.

Embodiment 3

[0037] Example 3: Using phage enzyme-linked immunoassay (ELISA) to screen specific single positive clones:

[0038] (1) From the cell culture dishes containing phage after the above 3-5 rounds of selection, select 96 single colonies and inoculate them in TB medium containing 100 micrograms per milliliter of ampicillin (1 liter of TB medium containing 2.3 grams of phosphoric acid Potassium dihydrogen, 12.52 g of dipotassium hydrogen phosphate, 12 g of peptone, 24 g of yeast extract, 4 ml of glycerol), after growth to the logarithmic phase, add a final concentration of 1 millimolar isopropyl thiogalactoside ( IPTG), cultivate overnight at 30-35°C.

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

[0040] (3) Wash the unbound antibody with PBST, add a mouse anti-HAtag antibody (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.), and place it at...

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PUM

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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 present invention belongs to the field of biomedicine or biopharmaceutical technology, and more specifically, relates to a nanobody (Nb68) directed against an epitope molecule of a CD105 polypeptide molecule, its coding sequence and use. 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 extracellular part contains 561 amino acids, and its transmembrane region contains 25 amino acids. Part of it contains 47 amino acids, which constitute the tail of CD105. In the extracellular part of CD105, there are 4 potential glycosylation sites, namely the 63rd, 96th, 10...

Claims

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Application Information

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