Feline herpes virus antibody sequence, tetrapeptide chain molecule and immunoglobulin molecule

A feline herpes virus, sequence technology, applied in the direction of antiviral immunoglobulin, immunoglobulin, peptide, etc.

Active Publication Date: 2020-10-30
青岛博隆基因工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The difficulty in solving the above problems and defects is as follows: in order to obtain an antibody library of a certain scale, the first thing to do is to use RT-PCR technology to obtain a complete set of antibody genes from the body. Verification of multiple pairs of primers for light and heavy chain variable region sequences

Method used

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  • Feline herpes virus antibody sequence, tetrapeptide chain molecule and immunoglobulin molecule
  • Feline herpes virus antibody sequence, tetrapeptide chain molecule and immunoglobulin molecule
  • Feline herpes virus antibody sequence, tetrapeptide chain molecule and immunoglobulin molecule

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Example 1 Construction of cat-derived anti-feline herpesvirus single-chain antibody library

[0063] 1. Isolation of Feline Peripheral Blood Lymphocytes

[0064] According to the International Animal Immunization Manual, 5 experimental cats were immunized with vaccine. Each immunization interval was 2 weeks, and a total of 3 immunizations were performed. Two weeks after the three immunizations, the peripheral blood of the cats was collected. Take 5-10mL of fresh blood and mix evenly with whole blood and tissue diluent at a ratio of 1:1-1:2. Add cell separation solution to a 15mL centrifuge tube, and gently add an equal volume of diluted anticoagulant blood along the tube wall. The horizontal centrifuge is centrifuged at a speed of 400g-800g and a time of 15min-25min. After centrifugation, the liquid in the centrifuge tube should be divided into four layers, from top to bottom: the first layer: plasma layer; the second layer: lymphocyte layer; The third layer: separa...

Embodiment 2

[0071] Example 2 Screening of cat-derived anti-feline herpesvirus single-chain antibody library

[0072] 1. Preparation of Phage Antibody

[0073] Add 1 mL of phage primary antibody library to 3 mL of XLI-Blue bacterial solution with OD600=0.5, place at 37°C for 45 min, add 6 mL of LB liquid medium containing Amp+ (100 μg / mL), and add glucose (1 mol / mL) at a ratio of 1:1000 L) Continue to cultivate, when the OD600 of the bacterial solution is 0.5, add the helper phage M13K07, place it in a 37°C incubator for 30min, and then incubate at 220rpm / min at 37°C for 1h, centrifuge the bacterial solution at 3500rpm for 10min, discard the supernatant, and use an equal volume LB liquid medium containing Amp+ (100 μg / mL) and Kana (50 μg / mL) was added with IPTG (1 mol / L) at a ratio of 1:1000, 220 rpm / min, 37°C, and shaken for 6 hours. Centrifuge the culture medium at 12000rpm for 10-20min, and collect the supernatant. Add PEG8000 at a ratio of 1:4-1:5, invert and mix well, at this time c...

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Abstract

The invention belongs to the technical field of virus antibodies, and discloses a feline herpes virus antibody sequence, a tetrapeptide chain molecule, an immunoglobulin molecule and an application thereof, and the sequences are a heavy chain variable region amino acid sequence SEQ ID NO: 1 and a nucleotide sequence SEQ ID NO: 3; and the amino acid sequence of the light chain variable region is SEQ ID NO: 2 and the nucleotide sequence is SEQ ID NO: 4. The sequence screening method comprises the following steps: preparing a bacteriophage antibody; screening the phage antibody library; identifying an anti-feline herpes virus feline phage single-chain antibody by using a PhageELISA method; sending the scFv bacterial liquid with a positive Phage ELISA identification result to a sequencing company for sequencing to obtain variable region sequences of a heavy chain and a light chain of the feline-derived anti-feline herpes virus genetic engineering antibody. The invention provides support for constructing a feline-derived anti-feline herpes virus genetic engineering antibody with high affinity and low immunogenicity. The product has important significance for promoting the development ofcat-derived antibody drugs.

Description

technical field [0001] The invention belongs to the field of virus antibody technology, and in particular relates to a virus antibody sequence, tetrapeptide chain molecule, immunoglobulin molecule and application. Background technique [0002] At present, feline herpesvirus type 1 (Feline herpesvirus 1, FHV-1) was isolated and identified for the first time in 1958 by Crandell and Maurer of the United States in local kittens suffering from respiratory diseases. The host of the virus was confirmed to be cats, but in Infection has also been found in cats such as lions and cheetahs. The transmission route of FHV-1 mainly includes the mouth, nose and conjunctiva to cause host infection, and its main latent site is the trigeminal nervous system. Clinically, FHV-1 is similar to FCV, and both can cause respiratory disease in cats. [0003] At present, the clinical treatment of feline herpes virus disease mainly relies on monoclonal antibodies. Most of the commercially available an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08G01N33/569C40B50/06
CPCC07K16/085C07K16/005G01N33/56983C40B50/06C07K2317/622C07K2317/56G01N2333/03
Inventor 曲雪婷尹燕博
Owner 青岛博隆基因工程有限公司
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