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Single domain heavy chain antibody capable of specifically combining c-Myc tag

A labeling and nanobody technology, applied in the field of genetically engineered antibodies, single domain heavy chain antibodies or polypeptides, can solve the problems of limited and complex sources of polyclonal antibodies, cumbersome development and production processes of monoclonal antibodies, etc.

Active Publication Date: 2017-07-14
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are monoclonal or polyclonal antibodies against c-Myc tag on the market for detection, but the development and production process of monoclonal antibodies is extremely cumbersome and complicated, and the sources of polyclonal antibodies are limited

Method used

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  • Single domain heavy chain antibody capable of specifically combining c-Myc tag
  • Single domain heavy chain antibody capable of specifically combining c-Myc tag

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Construction of an immune library of anti-c-Myc tag single domain heavy chain antibody (ie single domain heavy chain antibody against c-Myc tag)

[0024] The c-Myc tag was covalently coupled to bovine serum albumin (BSA) to obtain the c-Myc artificial antigen c-Myc-BSA. After emulsifying 300 μg of c-Myc-BSA with Freund's complete adjuvant, Alpacas (Lama pacos) were immunized by subcutaneous multipoint injection. For booster immunization, 150 μg c-Myc-BSA was emulsified with Freund's incomplete adjuvant at intervals of 2 weeks. Blood was collected from the vein 7 days after each immunization, and the serum titer was determined by indirect ELISA method. The sample with the highest serum titer was selected to separate lymphocytes. cells, RNA was extracted.

[0025] The extraction of RNA was carried out according to the instruction manual of RNAiso reagent from TAKARA company. Using RNA as a template and oligo dT as a primer, the first strand of cDNA was synthesized accor...

Embodiment 2

[0034] Panning and Identification of Anti-c-Myc Tag Single Domain Heavy Chain Antibody

[0035]The single domain heavy chain antibody against c-Myc tag was panned from the anti-c-Myc tag single domain heavy chain antibody immune library obtained in Example 1 by solid phase affinity panning. Add 120 μL of Myc-GST fusion protein (a protein fused with Myc tag and glutathione) diluted with PBS to each enzyme-labeled well, and coat overnight at 4°C. The coating concentration for each round of panning is 100, 75, 50 μg / mL; aspirate the coating solution, wash the plate 5 times with PBS, add 300 μL 3% BSA-PBS to each well, block for 2 hours at 37°C; wash the plate 5 times with PBS, add 100 μL phage antibody library (about 1×10 11 CFU), 37°C, incubate for 2.0 h; aspirate unbound phage, wash the plate with PBST (containing 0.5% Tween-20) for 3-5 times (increase 5 times for each round), and then wash the plate with PBS for 15-25 times; Use 100 μL eluent (glycine-hydrochloric acid, pH 2....

Embodiment 3

[0045] Scale Production of Anti-c-Myc Tag Single Domain Heavy Chain Antibody

[0046] Obtaining the DNA fragment encoding the anti-c-Myc tag single-domain heavy-chain antibody: 1. Using restriction endonuclease SfiI / NotI, double-digest the phagemid pHEN-anti-c-Myc single-domain heavy-chain antibody gene, and agar Glycogel electrophoresis to recover the anti-c-Myc tag single domain heavy chain antibody gene; 2. Directly send the anti-c-Myc tag single domain heavy chain antibody coding sequence to a biotechnology service company for chemical synthesis; 3. Design specific primers, through PCR technology amplifies from a cDNA library derived from alpaca (Lama pacos).

[0047] The obtained anti-c-Myc tag single domain heavy chain antibody gene fragment was cloned into the expression vector pET25-flag (the c-Myc tag carried by the vector itself was replaced with the Flag tag: DYKDDDDK), identified by PCR and enzyme digestion, and constructed Complete the E. coli expression plasmid ...

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Abstract

The invention belongs to the field of gene engineering and particularly provides a single domain heavy chain antibody which is direct at a c-Myc tag. The antibody has an amino acid sequence represented as the SEQ ID No.1 and is used in the fields of immunodetection, antigen enrichment and purification, etc. The amino acid sequence can be used as a precursor and modified through random or site-specific mutation technology, thereby producing a mutant, which has better properties (e.g., affinity, specificity, stability, etc.) for further development of proteins or polypeptides applied in the fields of medicines, industry and agriculture.

Description

technical field [0001] The present invention relates to single domain heavy chain antibody technology (also known as nanobody technology), and genetic engineering antibody technology, especially single domain heavy chain antibody or polypeptide directed at c-Myc tag. [0002] technical background [0003] The discovery of the c-Myc tagged protein originated in 1985 when Evan prepared a monoclonal antibody 9E10 against the human proto-oncogene product Myc protein. Later studies found that the epitope recognized by the antibody consists of 10 amino acid residues, and its sequence It is Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu, and these 10 amino acids can still maintain strong antigenic activity after fusion expression with other proteins, and can be recognized by corresponding antibodies without being affected by The effect of the protein framework. Therefore, the c-Myc tag system is widely used in the fields of immunological detection, cell imaging, affinity purification and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/32C12N15/13G01N33/68C07K1/22B01J20/24
CPCB01J20/24C07K1/22C07K16/32C07K2317/565C07K2317/567C07K2317/569G01N33/68G01N2333/82
Inventor 付金衡吴青松涂追许杨
Owner NANCHANG UNIV
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