Two scFv antibodies and coding genes thereof, and application of scFv antibodies to preparations for treatment or prevention of infectious bursal disease of chicken

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

Inactive Publication Date: 2018-09-04
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 can 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 antibodies and coding genes thereof, and application of scFv antibodies to preparations for treatment or prevention of infectious bursal disease of chicken
  • Two scFv antibodies and coding genes thereof, and application of scFv antibodies to preparations for treatment or prevention of infectious bursal disease of chicken
  • Two scFv antibodies and coding genes thereof, and application of scFv antibodies to preparations for treatment or prevention of 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, and 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 ...

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 primers composed of F1 and R1 to perform PCR amplification on the plasmid extracted from positive bacteria by flow cytometry screening as a template to obtain PCR amplification products.

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

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

[0051] 3. Digest the PCR amplified product of step 2 with restriction endonucleases NcoI and HindIII, and recover the digested product.

[0052] 4. Digest the pET-27b(+) vector with restriction endonucleases NcoI and HindIII to recover a vector backbone of about 5367bp.

[0053] 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 r...

Embodiment 3

[0062] Embodiment 3, the preparation of VP2 protein

[0063] 1. Construction of recombinant plasmids

[0064] 1. Synthesize the IBDV vp2 double-stranded DNA molecule shown in sequence 5 of the sequence listing.

[0065] 2. Using the double-stranded DNA molecule synthesized in the step as a template, the primers composed of F2 and R2 are used 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. The PCR amplified product of step 3 was double digested with restriction endonucleases BbsI and BamHI, and the digested product was recovered.

[0069] 4. Digest the plasmid pHisSUMO with restriction endonucleases BbsI and BamHI to recover a vector backbone of about 5700 bp.

[0070] 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 th...

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Abstract

The invention discloses two scFv antibodies and coding genes thereof, and application of the scFv antibodies to preparations for treatment or prevention of the infectious bursal disease of chicken. The invention provides two single-chain antibodies (i.e., the scFv antibodies). The scFv antibodies are capable of specifically binding to IBDV structural protein 2 (VP2) protein and a plurality of IBDVstrains, and can block the cytopathic effect (CPE) of IBDV on chicken embryo fibroblasts and protect young chickens infected with IBDV. Immune serum and egg yolk antibodies have the disadvantages ofcumbersome preparation, high production cost, unstable effect, difficulty in controlling of industrial production quality, incurrence of horizontally transmitted diseases and the like in use. The scFvantibodies provided by the invention can overcome the above disadvantages, has the advantages of strong specificity, good therapeutic effect, controllable industrial production quality and the like,avoids the problem of horizontally transmitted diseases caused by the egg yolk antibodies, and will create a new situation in the history of prevention and treatment of IBDV, or even in the whole history of prevention and treatment of animal viral 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] Chicken infectious bursal disease (Infectious Bursal Disease, IBD) is an acute, highly contagious chicken infectious disease caused by chicken infectious bursal disease virus (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 can also cause ...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/70C12N1/21G01N33/569A61K39/42A61P31/14
CPCA61K2039/505A61P31/14C07K16/10C07K2317/622C12N15/70G01N33/56983
Inventor 尹杰超李德山任桂萍郭笑辰魏颖孟婧刘铭瑶黄涛
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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