Immune library-derived nano-antibody for specifically recognizing immunoglobulin Fc fragment

A technology of immunoglobulin and nanobody, applied in the field of single domain heavy chain antibody, which can solve the problems of high production cost and cumbersome preparation process

Inactive Publication Date: 2016-12-07
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with single-domain heavy chain antibodies, traditional antibodies have disadvantages such as higher production costs and cumbersome preparation processes

Method used

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  • Immune library-derived nano-antibody for specifically recognizing immunoglobulin Fc fragment
  • Immune library-derived nano-antibody for specifically recognizing immunoglobulin Fc fragment
  • Immune library-derived nano-antibody for specifically recognizing immunoglobulin Fc fragment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Construction of Anti-Fc Single Domain Heavy Chain Antibody Immune Library

[0026] After emulsifying 300 μg of Fc recombinant protein (the protein can be obtained through commercial channels) with complete Freund's adjuvant, multi-point subcutaneous injection was performed to immunize alpacas (Lama pacos). 150 μg Fc recombinant protein was emulsified with Freund's incomplete adjuvant for booster immunization, and the interval was 2 weeks. Blood was collected from 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. Extract RNA.

[0027] 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 according to the instructions of TAKARA reverse transcriptase.

[0028] The gene encoding the variable region of...

Embodiment 2

[0036] Panning and Identification of Anti-IgG Fc Fragment Single Domain Heavy Chain Antibody

[0037] A solid-phase affinity panning method was used to pan out a single-domain heavy-chain antibody directed against the IgG Fc segment from an anti-Fc single-domain heavy-chain antibody immune library. The mouse serum was purified by affinity chromatography to obtain an IgG solution. Dilute the IgG solution with PBS to 50-100 μg / mL, add 100 μL to each well, and coat overnight at 4°C; suck out the coating solution, wash the plate with PBS 3 times, add 300 μL 3% BSA-PBS to each well, block for 2 hours at 37°C ; Wash the plate 6 times with PBS, add 100 μL phage antibody library (containing about 2×10 11 CFU), 37°C, incubate for 1.5 h; aspirate unbound phage, wash the plate with PBST (containing 0.5% Tween-20) 5 times (increase 1 time per round), and then wash the plate with PBS 10 times (the number of washes increases gradually 5 rounds); 100 μL of eluent (glycine-hydrochloric acid...

Embodiment 3

[0048] Expression and purification of anti-IgG Fc fragment single domain heavy chain antibody in Escherichia coli.

[0049] Acquisition of the DNA fragment encoding the anti-IgG Fc segment single-domain heavy chain antibody: 1. Use restriction endonuclease SfiI / NotI, double-digest the phagemid pHEN-X, and recover the anti-IgG Fc segment single domain by agarose gel electrophoresis Heavy chain antibody gene; 2. Directly send the anti-IgG Fc segment single domain heavy chain antibody coding sequence to a biotechnology service company for chemical synthesis; 3. Design specific primers and use PCR technology from the cDNA library derived from alpaca (Lama pacos) amplified in.

[0050] The obtained anti-IgG Fc-segment single-domain heavy-chain antibody gene fragment was cloned into the expression vector pRXS, identified by PCR and enzyme digestion, and the E. coli expression plasmid for the anti-IgG Fc-segment single-domain heavy-chain antibody was constructed, named pRXS-X.

[00...

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Abstract

The invention belongs to the field of gene engineering, and concretely relates to a single-domain heavy chain antibody for an immunoglobulin Fc fragment. The antibody has an amino acid sequence represented by SEQ ID NO:1, and can be used in the fields of immunodetection and antigen enrichment and purification. A mutant with good properties (endophilicity, specificity and stability) can be obtained through reconstructing by using a random or site directed mutagenesis technology with an amino acid sequence is used as a precursor, and is used for developing proteins or polypeptides for being further used in medicines, industries and the 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, in particular to the single domain heavy chain antibody against immunoglobulin G (immunoglobulin G, IgG) Fc segment (fragment crystalline, Fc) chain antibody or polypeptide. technical background [0002] Heavy-chain antibody (Heavy-chain antibody) is a kind of antibody that naturally lacks light chain and consists only of heavy chain, and exists in camels, sharks and other animals. Single-domain heavy chain antibody (also known as nanobody, VHH antibody, variable domain of heavy chain of heavy-chain antibody, the same below) refers to a genetically engineered antibody composed only of the variable region of heavy chain antibody (Variable region). Compared with ordinary antibodies, single domain heavy chain antibodies have the advantages of small molecular weight, high stability, and good water...

Claims

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Application Information

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IPC IPC(8): C07K16/00C12N15/13C07K1/22G01N33/68G01N33/53B01J20/24B01J20/281B01J20/30B01D15/38
CPCC07K16/005B01D15/3804B01J20/24B01J20/281B82Y30/00C07K2317/565C07K2317/567C07K2317/569G01N33/53G01N33/6854
Inventor 涂追付金衡许杨周鹏
Owner NANCHANG UNIV
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