Application of scFv (single-chain fragment variable) antibody for resisting infectious bursal disease viruses to preparation for treating or preventing infectious bursal disease

A single-chain antibody and active technology, applied in the direction of virus/bacteriophage, anti-viral immunoglobulin, application, etc., can solve the problems of poor controllability and limitations in industrial production, and achieve the avoidance of horizontal transmission of diseases, strong specificity, and therapeutic effect Good results

Active Publication Date: 2016-04-20
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 (single-chain fragment variable) antibody for resisting infectious bursal disease viruses to preparation for treating or preventing infectious bursal disease
  • Application of scFv (single-chain fragment variable) antibody for resisting infectious bursal disease viruses to preparation for treating or preventing infectious bursal disease
  • Application of scFv (single-chain fragment variable) antibody for resisting infectious bursal disease viruses to preparation for treating or preventing infectious bursal disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Discovery of scFv-GXC 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, induc...

Embodiment 2

[0037] Embodiment 2, preparation of scFv-GXC 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. Digest the PCR amplified product in step 2 with restriction endonucleases NdeI and HindIII, and recover the digested product.

[0043] 4. Digest the pET-27b(+) vector with restriction endonucleases NdeI and HindIII to recover a vector backbone of about 5367 bp.

[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 is described as follows: a double-stranded DNA molecule shown ...

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. The PCR amplified product of step 2 was double-digested with restriction endonucleases BbsI and BamHI, and the digested product was recovered.

[0060] 4. Digest the plasmid pHisSUMO with restriction endonucleases BbsI and BamHI to recover a vector backbone of about 5700 bp.

[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 coding sequence of the VP2 protein coding g...

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Abstract

The invention discloses application of an scFv (single-chain fragment variable) antibody for resisting infectious bursal disease viruses to a preparation for treating or preventing infectious bursal disease. The provided svFv-XC antibody is composed of a heavy chain variable region, a light chain variable region and a connecting region between the heavy chain variable region and the light chain variable region, wherein the heavy chain variable region is the following (a) or (b), (a) is protein composed of amino acid residues from 1<st> to 123 in the sequence 1, and (b) is protein which is obtained by substituting and / or missing and / or adding the amino acid residues and has the same activity; the light chain variable region is the following (c) or (d), (c) is protein composed of amino acid residues from 139 to 244 in the sequence 1, and (d) is protein which is obtained by substituting and / or missing and / or adding the amino acid residues and has the same activity. The provided svFv-XC antibody is obtained through screening by using an antigen-antibody coexpression bacterial display technology, the neutralization activity of the svFv-XC antibody is higher than that of an svFv-D antibody granted with the patent right already by 60 times or more, and the svFv-XC antibody has the advantages of being high in specificity, better in treatment effect and the like.

Description

technical field [0001] The invention relates to the application of scFv antibody against chicken infectious bursal virus in treating or preventing chicken infectious bursal disease preparation. Background technique [0002] Chicken infectious bursal disease (Infectious Bursal Disease, IBD) is an acute, highly contagious infectious disease caused by chicken infectious bursal disease virus (Infectious Bursal Disease Virus, IBDV). Young chickens can damage the central immune organ of chickens - the bursa of Fabricius. It has the characteristics of fast transmission speed, strong infectivity, high infection rate and high mortality rate. The disease is currently distributed worldwide and is one of the most important diseases in 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 susceptibilit...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N7/00G01N33/569
CPCA61K2039/505C07K16/10C07K2317/622C12N7/00C12N2720/10022
Inventor 李德山郭笑辰任桂萍曹宏雪
Owner JIANGSU KANIONREAL BIOMEDICAL TECH CO LTD
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