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Two ScFv (Single Chain Variable Fragment ) antibodies, encoding genes and application thereof for preparing preparation for treating or preventing infectious bursal disease of chicken

An ibdv-vp2scfv29, coding technology, applied in antiviral agents, applications, antibodies, etc., can solve the problems of vaccine immunization failure, bacterial secondary infection, poor controllability, etc., to achieve strong specificity, good therapeutic effect, and avoidance level. The effect of spreading disease

Inactive Publication Date: 2014-01-01
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is very easy to cause secondary infection of bacteria (Escherichia coli and Salmonella), and it may also cause immune failure of other vaccines
Polyclonal antibody (hyperimmune serum and egg yolk antibody) is an effective treatment at present, and the treatment effect is good in the early stage of the disease. leukemia) and allergic reactions caused by some non-specific antibodies

Method used

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  • Two ScFv (Single Chain Variable Fragment ) antibodies, encoding genes and application thereof for preparing preparation for treating or preventing infectious bursal disease of chicken
  • Two ScFv (Single Chain Variable Fragment ) antibodies, encoding genes and application thereof for preparing preparation for treating or preventing infectious bursal disease of chicken
  • Two ScFv (Single Chain Variable Fragment ) antibodies, encoding genes and application thereof for preparing preparation for treating or preventing infectious bursal disease of chicken

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Discovery of scFv antibody (single-chain antibody) and its coding gene

[0039] 1. Construction of antibody library

[0040] 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 pBSD to construct a bacterial surface display library of anti-IBDV antibodies. VH is about 380bp, VL is about 320bp, VH-Tlinker-VL is about 740bp;

[0041] 2. Antibody library screening

[0042] Collect all clones after transformation, induce with IPTG (0.25mmol / ml) for 4h, and pass...

Embodiment 2

[0046] Embodiment 2, preparation of scFv antibody

[0047] 1. Synthesize the double-stranded DNA molecule shown in sequence 2 of the sequence listing;

[0048] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, use the primers composed of F1 and R1 to perform PCR amplification on the plasmid extracted from the positive bacteria by flow cytometry screening as a template to obtain a PCR amplification product;

[0049] F1: 5'–CATG CCATGG GCCGTGACGTTGGACGAG-3';

[0050] R1: 5'-CCC AAGCTT TTAACCTAGGACGGTCAGGG-3;

[0051] 3. Using restriction endonucleases Nco I and Hind III double enzyme digestion of the PCR amplification product of step 2, reclaiming the enzyme digestion product;

[0052] 4. Using restriction endonucleases Nco I and Hind III Digest the pET-27b(+) vector with double enzymes, and recover the vector skeleton of about 5367bp;

[0053] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain a reco...

Embodiment 3

[0062] Embodiment 3, the preparation of VP2 protein

[0063] 1. Construction of recombinant plasmids

[0064] 1. Synthesizing the IBDV vp2 double-stranded DNA molecule shown in sequence 6 of the sequence listing;

[0065] 2. Using the double-stranded DNA molecule synthesized in the step as a template, use the primers composed of F2 and R2 to perform PCR amplification on vp2 to obtain a PCR amplification product;

[0066] F2: 5'- GAAGAC TTAGGT ACAAACCTGCAAGATCAA-3';

[0067] R2: 5'- GGATCC TTATGCTCCTGCAATCTTCAG-3';

[0068] 3. Using restriction endonucleases Bbs I and Bam H 1 double enzyme digestion of the PCR amplification product of step 3, reclaiming the enzyme digestion product;

[0069] 4. Using restriction endonucleases Bbs I and Bam H I double-digest the plasmid pHisSUMO, and recover the vector backbone of about 5700bp;

[0070] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain a recombinant plasmid. In the recombinant...

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Abstract

The invention discloses two types of ScFv (Single Chain Variable Fragment) antibodies, encoding genes of the antibodies and applications of the antibodies for preparing the preparations for treating or preventing infectious bursal disease of chicken. Two single-chain antibodies (namely, the ScFv antibodies) are provided; and the ScFv antibodies can be specially bound with the protein 2 (VP2) (Virus Protein 2) in an IBDV (Infectious Bursal Disease Virus) structure and a plurality of IBDV strains to block the cytopathic effect (CPE) of the chicken embryo fibroblast by the IBDV so as to protect the young chicken infected with IBDV. The immune serum and egg yolk antibody are tedious in preparation, high in production cost, unstable in effect, difficult to control industrialized production quality, capable of causing horizontal disease spread and the like in a use process; the ScFv antibodies disclosed by the invention can overcome the above disadvantages and have the advantages of strong specificity, good treatment effect, controllable industrialized production quality, capability of preventing the horizontal disease spread caused by the egg yolk antibody and the like; and therefore, by virtue of the ScFv antibodies, a new situation is opened up in the IBDV prevention and treatment history, and even in the entire prevention and treatment history for animal virus diseases.

Description

technical field [0001] The invention relates to two kinds of scFv antibodies, their coding genes and their application in preparing preparations for treating or preventing chicken infectious bursal disease. Background technique [0002] Infectious Bursal Disease (IBD) is an acute and highly contagious chicken infectious disease caused by Infectious Bursal Disease Virus (IBDV). It is characterized by strong infectivity, high infection rate and high mortality rate. The disease causes significant economic losses to the poultry industry. [0003] IBDV can multiply rapidly in the lymphocytes in the chick's bursa, especially the B lymphocytes, prompting the apoptosis of the B lymphocytes, and the extreme atrophy of the bursa, eventually leading to immunodeficiency and immunosuppression. Therefore, it is very easy to cause secondary infection of bacteria (Escherichia coli and Salmonella), and it can also cause immune failure of other vaccines. Polyclonal antibody (hyperimmune se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/70C12N1/21G01N33/569A61K39/42A61P31/14
Inventor 尹杰超周兵李天鹤李德山张瑛杰张立夏
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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