A nanobody against green fluorescent protein, applications and GFP immunoaffinity adsorption material

A technology of green fluorescent protein and nano-antibody, which is applied in the application, analysis of materials, material inspection products, etc., can solve the problems of poor antibody stability, high production cost, and complexity, and achieve simple production process, small molecular weight, and high specificity Effect

Active Publication Date: 2021-02-02
北京兰博利德商贸有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, most of the monoclonal or polyclonal antibodies against GFP are used for detection in the market, but the development and production process of monoclonal antibodies is extremely cumbersome and complicated, and the antibody stability is poor, the production cost is high, and the source of polyclonal antibodies is limited
Since traditional antibodies contain Fc segments, they are prone to non-specific binding or contamination

Method used

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  • A nanobody against green fluorescent protein, applications and GFP immunoaffinity adsorption material
  • A nanobody against green fluorescent protein, applications and GFP immunoaffinity adsorption material
  • A nanobody against green fluorescent protein, applications and GFP immunoaffinity adsorption material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Construction of a Nanobody library for GFP:

[0062] (1) The concentration of GFP is 500 micrograms per milliliter. For each immunization, mix 1 mg of GFP with Freund's adjuvant in an equal volume to immunize a Xinjiang Bactrian camel once a week for a total of 4 times. Freund's adjuvant, and Freund's incomplete adjuvant was used for the remaining several times to stimulate B cells to express antigen-specific nanobodies during the immunization process.

[0063] (2) After the 4 times of immunization, extract 100 ml of camel peripheral blood lymphocytes and extract total RNA, referring to the RNA extraction kit provided by QIAGEN.

[0064] (3) According to the instructions of Super-Script III FIRST STRANDSUPERMIX kit, the extracted RNA was reverse-transcribed into cDNA and the VHH chain was amplified by nested PCR. The first round of PCR:

[0065] Upstream primer: GTCCTGGCTGCTCTTCTACAAGGC

[0066] Downstream primer: GGTACGTGCTGTTGAACTGTTCC

[0067] Amplify t...

Embodiment 2

[0075] Embodiment 2: Nanobody screening process against GFP:

[0076] (1) Dissolve in 100mM pH 8.2NaHCO 3 200 micrograms of GFP in the medium were coupled to a microtiter plate, placed overnight at 4°C, and a negative control was set up at the same time.

[0077] (2) On the second day, 100 microliters of 0.1% casein were added to the two wells respectively, and blocked for 2 hours at room temperature.

[0078] (3) After 2 hours, add 100 μl phage (8*10 11 tfu immunized camel nanobody phage display gene library) and acted for 1 hour at room temperature.

[0079] (4) Wash 5 times with PBST (PBS containing 0.05% Tween 20) to wash away unbound phages.

[0080] (5) Use triethylamine (100mM) to dissociate the phage that specifically binds to GFP, and infect Escherichia coli TG1 that is in logarithmic growth, produce and purify the phage for the next round of screening, the same screening process Repeat for 3-4 rounds. In the process of continuous screening, positive clones will ...

Embodiment 3

[0081] Embodiment 3: use the enzyme-linked immunosorbent method (ELISA) of phage to screen specificity single positive clone:

[0082] (1) From the cell culture dish containing phage after the above 3-4 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 IPTG with a final concentration of 1 millimolar, and cultivate overnight at 28 ° C.

[0083] (2) Obtain the crudely extracted antibody by infiltration method, transfer the antibody to an antigen-coated ELISA plate, and place it at room temperature for 1 hour.

[0084] (3) Unbound antibodies were washed away with PBST, a mouse anti-HAtag antibody (anti-mouse anti-HA antibody, purchased from Beijing Kangwe...

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Abstract

The present invention relates to a nanobody against green fluorescent protein, and the antibody has the amino acid sequence shown in SEQ ID NO.1. The nanobody is a single-domain antibody heavy chain antibody (nanobody) that can specifically bind to GFP, and can be used for the detection and purification of GFP and GFP fusion proteins, such as the preparation of reagents and tools for the detection and purification of GFP Wait.

Description

technical field [0001] The invention belongs to the field of single-domain heavy chain antibody technology (also known as nanobody technology) and genetic engineering antibody technology, in particular to a nanobody for green fluorescent protein (GFP), its application and GFP immunoaffinity adsorption material. Background technique [0002] GFP is a protein composed of about 238 amino acids, which can be excited by blue light to ultraviolet light and emit green fluorescence. GFP is widely used in immunological detection, cell imaging, affinity purification and protein engineering and other fields. [0003] At present, most of the monoclonal or polyclonal antibodies against GFP are used for detection on the market, but the development and production process of monoclonal antibodies is extremely cumbersome and complicated, and the antibody stability is poor, the production cost is high, and the source of polyclonal antibodies is limited. Since traditional antibodies contain F...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/13G01N33/533G01N33/68
CPCC07K16/18G01N33/533G01N33/68
Inventor 董春明
Owner 北京兰博利德商贸有限公司
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