Application of scFv antibody in preparation used for treatment or prevention of infectious bursal disease of chicken

A single-chain antibody and active technology, applied in antiviral agents, applications, antibodies, etc., can solve the problems of poor controllability in industrial production, and achieve the effects of avoiding horizontal transmission of diseases, strong specificity, and good therapeutic effect

Active Publication Date: 2016-03-09
JIANGSU KANIONREAL BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibody drugs are currently effective therapeutic drugs. Hyperimmune serum and egg yolk antibodies can play a good role in the early stage of the disease, but they are limited due to poor controllability of industrial production and the existence of horizontal transmission of diseases.

Method used

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  • Application of scFv antibody in preparation used for treatment or prevention of infectious bursal disease of chicken
  • Application of scFv antibody in preparation used for treatment or prevention of infectious bursal disease of chicken
  • Application of scFv antibody in preparation used for treatment or prevention of infectious bursal disease of chicken

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Discovery of scFv-GD antibody (single-chain antibody) and its coding gene

[0031] 1. Construction of antibody library

[0032]The spleens of chickens immunized with IBDV were taken, RNA was extracted and reverse transcribed into cDNA. According to the antibody sequence on GenBank, gene primers for cloning the variable region of the antibody were designed, and cDNA was used as a template to clone the variable region of the antibody by PCR. The VH and VL fragments were respectively inserted into the upstream and downstream of the Linker of the pTlinker vector to construct a scFv antibody library. The scFv antibody library was digested and connected to the bacterial display vector pBFD-Ab-VP2 to construct a bacterial surface display library co-expressed with anti-IBDV antigen antibody. VH is about 380bp, VL is about 320bp, and VH-Tlinker-VL is about 740bp.

[0033] 2. Antibody library screening

[0034] All clones after transformation were collected, induce...

Embodiment 2

[0037] Embodiment 2, preparation of scFv-GD antibody

[0038] 1. Synthesize the double-stranded DNA molecule shown in sequence 2 of the sequence listing.

[0039] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of F1 and R1 to obtain a PCR amplification product.

[0040] F1: 5'–CGC CATATG GCCGTGACGTTGGACGAG-3';

[0041] R1: 5'–CCC AAGCTT TTAACCTAGGACGGTCAGGG-3'.

[0042] 3. Using restriction endonucleases Nde I and Hind III Double enzyme digestion of the PCR amplification product of step 2, and recovery of the enzyme digestion product.

[0043] 4. Using restriction endonucleases Nde I and Hind III double-digested the pET-27b(+) vector, and recovered the vector backbone of about 5367bp.

[0044] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain a recombinant plasmid. According to the sequencing results, the structure of the recombinant plasmid...

Embodiment 3

[0053] Embodiment 3, the preparation of VP2 protein

[0054] 1. Construction of recombinant plasmids

[0055] 1. Synthesize the double-stranded DNA molecule shown in sequence 4 of the sequence listing.

[0056] 2. Using the double-stranded DNA molecule synthesized in the step as a template, perform PCR amplification with a primer pair composed of F2 and R2 to obtain a PCR amplification product.

[0057] F2: 5'- GAAGAC TTAGGTACAAACCTGCAAGATCAA-3';

[0058] R2: 5'- GGATCC TTATGCTCCTGCAATCTTCAG-3'.

[0059] 3. Using restriction endonucleases Bbs I and Bam H I double enzyme digestion of the PCR amplification product of step 2, and reclaim the enzyme digestion product.

[0060] 4. Using restriction endonucleases Bbs I and Bam H I double-digested the plasmid pHisSUMO, and recovered the vector backbone of about 5700bp.

[0061] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain a recombinant plasmid. In the recombinant plasmid, the ...

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PUM

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Abstract

The invention discloses application of an scFv antibody in a preparation used for treatment or prevention of the infectious bursal disease of chicken. The scFv-GD antibody provided by the invention comprises a heavy-chain variable region, a light-chain variable region and a joining region between the heavy-chain variable region and the light-chain variable region. The heavy-chain variable region is (a) or (b), wherein (a) is protein composed of the first to the 128th amino acid residues in a sequence 1, and (b) is protein derived from (a) through substitution and/or deletion and/or addition of amino acid residues and having same activity. The light-chain variable region is (c) or (d), wherein (c) is protein composed of the 144th to the 249th amino acid residues in the sequence 1, and (d) is protein derived from (c) through substitution and/or deletion and/or addition of amino acid residues and having same activity. The scFv-GD antibody is obtained through screening via antigen-antibody co-expression bacterium display techniques; the neutralization activity of the scFv-GD antibody is more than 10 times of the neutralization activity of a patented scFv-A antibody; and the scFv-GD antibody has the advantages of good specificity and better treatment effect.

Description

technical field [0001] The invention relates to the application of a scFv antibody in a preparation for treating or preventing chicken infectious bursal disease. Background technique [0002] Chicken infectious bursal disease (Infectious Bursal Disease, IBD) is caused by chicken infectious bursal disease virus (Infectious Bursal Disease Virus, IBDV), an acute and highly contagious infection characterized by the destruction of chicken immune central organ - bursa It has the characteristics of fast transmission, strong infectivity, high infection rate and high mortality rate. The disease has spread all over the world and is one of the main diseases that endanger the poultry industry, and the economic loss caused by the failure of immunity is huge. [0003] IBDV can multiply rapidly in lymphocytes in chick bursa, especially B lymphocytes, resulting in immunosuppression, which can increase the body's susceptibility to other pathogens and reduce the responsiveness to other vacci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13G01N33/569A61K39/42A61P31/14
Inventor 李德山郭笑辰任桂萍
Owner JIANGSU KANIONREAL BIOMEDICAL TECH CO LTD
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