Avian infectious bronchitis virus (IBV)-like particle and preparation method and application thereof

A bronchitis and virus-like technology, applied in the fields of genetic engineering and medical immunology, can solve the problem of few veterinary VLPs vaccines, achieve good innate immunity and mucosal immune response, strong immune protection, and good immune effect

Active Publication Date: 2021-04-09
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it has been reported that more than 100 virus VLPs have been successfully constructed, there are very few commercialized veterinary VLPs vaccines, and there is no VLPs vaccine for IB on the market. The construction of VLPs vaccines has great commercial value and development potential

Method used

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  • Avian infectious bronchitis virus (IBV)-like particle and preparation method and application thereof
  • Avian infectious bronchitis virus (IBV)-like particle and preparation method and application thereof
  • Avian infectious bronchitis virus (IBV)-like particle and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Preparation and identification of embodiment 1 recombinant S, M, E protein

[0040] FastBac TM / HBM-TOPO transposable vector and DH10Bac TM Competent cells were purchased from Invitrogen; Sf9 insect cells and IBV GX-YL5 strain were preserved by the Institute of Poultry Breeding and Poultry Diseases, Guangxi University.

[0041] 1) Primer design and synthesis

[0042] Refer to the IBV GX-YL5 complete gene sequence included in NCBI GenBank (GenBank serial number: HQ848267.1), find out the corresponding S, M and E gene sequences, and use the SignalP4.1 Server online software to analyze the signal peptides of the three genes respectively. Prediction, after removing the signal peptide sequence, start codon and stop codon (there are start codon and stop codon on the carrier), design primers corresponding to the 3 genes and M13 for later PCR identification of recombinant bacmid and post-transfection PCR identification The universal primers for the vector were synthesized by...

Embodiment 2

[0149] Preparation and identification of embodiment 2 SME-VLPs

[0150] 1) Preparation of SME-VLPs

[0151] Under the optimal conditions of exploration, press 2×10 6 cells / mL inoculated Sf9 cells (vitality > 90%) in cell suspension culture flasks, and after suspension culture in a constant temperature incubator at 27°C for 12 hours, the cells grew stably. According to the same MOI (S:M:E= 3:3:3) co-infect the Sf9 insect cells in suspension culture, and harvest the cell suspension at 84h.

[0152] 2) Sucrose density gradient centrifugal purification of SME-VLPs

[0153] The above harvested cell suspension was centrifuged at 4°C and 2000r / min for 20min, the supernatant was discarded, the cell pellet was collected and resuspended in pre-cooled PBS, and then placed at -80°C for three times of repeated freezing and thawing, and then ultrasonicated on ice ( Work for 5s, rest for 5s) to fully release the virus particles, sonicate until it is no longer viscous, then the crushing is...

Embodiment 3

[0161] Immunogenicity and immune protection research of embodiment 3SME-VLPs

[0162] Preparation of oil-emulsion seedlings

[0163] (1) The inactivated GX-YL5 allantoic fluid is made into an aqueous phase by containing 2 μg S protein / feather volume ratio, virus liquid: autoclaved Tween-80=96:4, No. 10 white oil: Siben- 80=10:1 mix well, make oil phase after autoclaving. The water phase is added dropwise to the oil phase (water phase: oil phase = 2:3), fully oscillated evenly, and the inactivated oil emulsion seedlings of the water-in-oil property are made;

[0164] (2) SME-VLPs containing 2 μg of S protein / feather were completely emulsified and mixed with an equal amount of Freund's complete adjuvant to prepare an oil-free emulsion vaccine. Completely emulsify and mix with Freund's incomplete adjuvant according to the same dose, and prepare the second free oil emulsion vaccine.

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Abstract

The invention discloses an avian infectious bronchitis virus (IBV)-like particle. The virus-like particle is prepared by the following steps of: transferring IBV-S, IBV-M and IBV-E genes into a baculovirus expression system respectively to obtain three recombinant rod particles; transfecting the three recombinant rod particles into insect cells respectively to obtain recombinant baculovirus rHBM-S, rHBM-M and rHBM-E; and then carrying out primary construction through a co-infection form. The virus-like particle is applied to preparation of vaccines and has a great development potential.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and medical immunology, and more specifically relates to an avian infectious bronchitis virus-like particle and a preparation method and application thereof. Background technique [0002] Chicken infectious bronchitis (Infectious bronchitis, IB) is an acute and highly contagious infectious disease of chickens caused by coronavirus chicken infectious bronchitis virus (Infectious bronchitis virus, IBV), which is stipulated by the International Bureau of Epizootics and my country. One of the second-class infectious diseases of poultry. Chickens of different ages, sexes and breeds were susceptible to IB. The harm of the disease is mainly in three aspects: first, it destroys the integrity of the respiratory mucosa, promotes the occurrence of other conditional diseases such as mycoplasma mixed infection and secondary infection such as Escherichia coli, which increases the mortality of chick...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C12N15/50C12N15/866A61K39/215A61P31/14
CPCC07K14/005C12N15/86A61K39/12A61P31/14C12N2770/20022C12N2770/20023C12N2770/20034C12N2710/14043A61K2039/5258Y02A50/30
Inventor 磨美兰张愉袁园张丽华范文胜韦兰萍韦平韦天超黄腾
Owner GUANGXI UNIV
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