Recombinant Newcastle disease vector vaccine for expressing avian infectious bronchitis virus S protein, preparation method and application

A technology of bronchitis and carrier vaccines, applied in the biological field, can solve the problems of poultry breeding industry loss, kidney and respiratory damage, etc., and achieve the effect of improving protection efficiency

Active Publication Date: 2021-10-01
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Infectious bronchitis virus (infectious bronchitis virus, IBV) infection causes poultry acute highly contagiou

Method used

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  • Recombinant Newcastle disease vector vaccine for expressing avian infectious bronchitis virus S protein, preparation method and application
  • Recombinant Newcastle disease vector vaccine for expressing avian infectious bronchitis virus S protein, preparation method and application
  • Recombinant Newcastle disease vector vaccine for expressing avian infectious bronchitis virus S protein, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Modification of chicken infectious bronchitis virus (IBV) S gene and construction strategy of expressing different forms of S protein and fusion Nd peptide recombinant NDV infectious cDNA clone

[0047] The total length of the S gene of infectious bronchitis virus IBV QX-like strain is 3,471 bp, and the coding region encodes 1156 amino acids. The mature S protein is composed of S1 subunit and S2 subunit produced by the cleavage of its precursor. Among them, the S1 protein contains 554 amino acids with a size of about 60.7ku, and the S2 protein contains 606 amino acids with a size of about 66.6ku.

[0048] In this application, by analyzing the signal peptide of the S protein, the results are as follows figure 1 It is shown in the figure that the 20 amino acids at the N-terminal of the S protein are the signal peptide sequence of the S protein, and primers are designed and synthesized to amplify the S protein sequence respectively, which are the full length of t...

Embodiment 2 4

[0051] Example 2 Construction of four recombinant Newcastle disease vector vaccine infectious cDNA cloning plasmids

[0052] Design primers according to the gene sequence of pTS09-C, reversely amplify the entire plasmid from the M gene of NDV to the full length, and add 15-25 bp of the inserted gene to the 5' end of the upstream primer and the 5' end of the downstream primer respectively. For the source arm, the exogenous insert fragments S, S1, tS1, and tS1 / Nd were also amplified using primers containing homology arms to construct plasmids pTS-S, pTS-S1, pTS-tS1, and pTS-tS1 / Nd, respectively. See Table 1 for the sequence.

[0053] Table 1 Primers used to construct plasmids pTS-S, pTS-S1, pTS-tS1 and pTS-tS1 / Nd

[0054]

[0055] Using the pTS09-C plasmid, high-fidelity enzymes and corresponding primers were used for PCR amplification, and the linearized target vector fragment was obtained, with a size of about 17,000 bp, which was in line with expectations. At the same ti...

Embodiment 3 4

[0064] Example 3 Rescue and Identification of Four Recombinant Newcastle Disease Vector Vaccine Viruses

[0065] Transform the helper plasmids required for transfection and the recombinant pTS-S, pTS-S1, pTS-tS1 and pTS-tS1 / Nd plasmids into E.coli DH5α respectively, spread the resistant LB agar plate, and culture overnight at 37°C. After the positive colonies were selected and inoculated into LB liquid medium for shaking, the plasmid was extracted using the endotoxin-free plasmid extraction kit for subsequent transfection experiments.

[0066] Insert BHK-21 cells into a 6-well plate, culture for 12 hours, and transfect after 2 hours of infection with poxvirus expressing T7 RNA polymerase; use Opti-MEM medium to dilute Lipofectamine 3000 reagent, and recombinant pTS-S, pTS -S1, pTS-tS1, and pTS-tS1 / Nd plasmids were mixed with helper plasmids to ensure that the total amount of DNA was 2 μg, and then added to Opti-MEM medium, and added P3000 TM Reagent, mix thoroughly; mix DNA a...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a recombinant Newcastle disease vector vaccine for expressing avian infectious bronchitis virus S protein, a preparation method and application. According to the invention, a reverse genetic manipulation technology is adopted to insert an IBV S protein coding gene into a thermally stable Newcastle disease vaccine strain (rTS09-C) genome in different forms, and a recombinant thermally stable Newcastle disease vector vaccine strain for expressing IBV S proteins in three forms and a recombinant Newcastle disease vector vaccine strain for expressing the S protein and N protein T cell epitope conserved peptide fragment in an optimized form are obtained through constructing and screening. The four constructed recombinant thermally-stable Newcastle disease vector vaccine strains for expressing IBV antigen protein have the characteristics of heat resistance, low toxicity and stable hereditary characteristic; and the immunization of the recombinant Newcastle disease vector vaccine can induce the chicken body to generate specific immune response and immune protection against lethal challenge infection of NDV and IBV virulent strains.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant Newcastle disease vector vaccine expressing the S protein of avian infectious bronchitis virus, a preparation method and application. Background technique [0002] Currently, the two most serious diseases to poultry farming are Newcastle disease (ND) and highly pathogenic avian influenza (HPAI). In addition, because the scope of influence of infectious bronchitis (infectious bronchitis, IB) in chickens is gradually expanding, people have paid great attention to this poultry virus in recent years. Newcastle disease, highly pathogenic avian influenza and chicken infectious bronchitis are caused by Newcastle disease virus (Newcatledisease virus, NDV), highly pathogenic avian influenza virus (high pathogenic avian influenza virus, HPAIV) and infectious bronchitis virus (Infectious bronchitis virus, IBV) is a severe infectious disease caused by infecting poultry...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/50A61K39/295A61K39/17A61K39/215A61P31/14C12R1/93
CPCC12N7/00C07K14/005A61K39/12A61P31/14C12N2760/18121C12N2760/18134C12N2760/18143C12N2770/20022C12N2770/20034A61K2039/5256A61K2039/552A61K2039/70Y02A50/30
Inventor 潘兹书许路来温国元
Owner WUHAN UNIV
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