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Avian influenza and infectious bronchitis hybrid virus-like particle as well as preparation method and application thereof

A technology for influenza virus and bronchitis, applied in the field of mixed virus-like particles of avian influenza and infectious bronchitis, which can solve the problems of live virus leakage and diffusion, long production cycle and high production conditions, and achieve good immune effect, long duration, The effect of strong immunity

Inactive Publication Date: 2012-03-14
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, the distinctive characteristics of influenza viruses, such as high frequency of mutations, recombination of genetic material and antigenic drift between dual and multiple subtype viruses, have greatly challenged the long-term safety and effectiveness of these vaccines. Even "ineffective vaccines" have been produced, which are difficult to cope with the infection of new mutant influenza viruses
In addition, these vaccine preparations also have the following shortcomings: (1) Long production cycle: it takes 5-8 months from obtaining new subtype virus strains to vaccine production and marketing, and it is difficult to contain new outbreaks
(2) High production conditions: In order to prevent artificial pollution of the environment and the leakage and spread of the live virus produced, the whole set of production must be carried out under the conditions of a biosafety level 3 laboratory according to regulations, and the inactivation of the virus must be completely and thoroughly
(3) Inability to distinguish vaccinated chickens from virus-infected chickens
Now it has been determined that mRNA A, mRNA C, and mRNA E encode three main structural proteins of IBV: nucleocapsid protein (N protein), matrix protein (Membrane glycoprotein, M protein), and spike protein (spike glycoprotein, S protein); mRNA B and D encode 7.5KD, 9.5KD and 6.7KD, 7.4KD, 12.4KD, respectively, five small proteins whose functions are still unclear; and mRNA F may encode RNA-dependent RNA polymerase, However, direct evidence is lacking

Method used

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  • Avian influenza and infectious bronchitis hybrid virus-like particle as well as preparation method and application thereof
  • Avian influenza and infectious bronchitis hybrid virus-like particle as well as preparation method and application thereof
  • Avian influenza and infectious bronchitis hybrid virus-like particle as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0057] Embodiment 2: Construction expresses the insect baculovirus expression plasmid of M1, NP, HA, NA, S / HA gene and in insect cell synthetic recombinant insect baculovirus

[0058] (1) Construction of recombinant plasmids expressing M1 and NP genes

[0059] Insect baculovirus plasmid PFastBac (product of Invitrogen Company) was digested with restriction endonuclease Sal I / Hind III at 37°C for 3 hours, and the digested plasmid PFastBac was recovered and purified with a gel recovery kit. Under the action of T4 DNA ligase, the digested plasmid and the digested M1 DNA fragment were ligated overnight at 16°C. The reaction system is as follows: 1ml of 10×T4 ligation buffer, 3ml of DNA fragment recovered by M1 digestion, 1 μl of product recovered from PFastBac plasmid digestion, 1 μl of T4 DNA ligase, ddH 2 O to make up to 10 μl. Use the heat shock method to transfer the ligation product into Top10 competent cells, add it to a small plastic centrifuge tube, mix gently, place...

Embodiment 3

[0075] Example 3: Expression of M1, NP, HA, S / HA and NA genes in co-transfected suspension cultured insect cells sf-9

[0076] 200ml sf-9 cell mixture suspension culture is placed in the Erlenmeyer shaker flask of 1 liter of volumes, and cell culture fluid is the sf-900II (or the Grace insect culture medium of Invitrigen Company) without serum, and the shaking speed of shaker is 100rpm, The temperature was kept constant at 27°C. When the cell concentration reaches 2×10 6 At cells / ml, sf9 cells were co-transfected with Bac-M1, Bac-NP, Bac-HA-NA, Bac-S / HA-NA insect baculovirus. The MOI ratio of the virus was 3 (Bac-M1 and Bac-NP): 1 (Bac-HA-NA): 1 (Bac-S / HA-NA). After the transfected cells were shaken and cultured at constant temperature for 3 days, all samples were collected, centrifuged at 4°C for 30 minutes at a spin speed of 3000 rpm, and the supernatant was collected. The centrifuged cell pellet was treated with cell lysate and centrifuged at 4°C for 10 minutes at a sp...

Embodiment 4

[0077] Example 4: Purification of virus-like particles, indirect immunofluorescence detection and electron microscope observation.

[0078] Put the cell supernatant collected by the above centrifugation into a 13ml ultracentrifuge tube, weigh, balance, and seal the tube, put it into an ultracentrifuge (product of Bechmem Company), centrifuge at 100,000rpm at 4°C for 1 hour, and then take out the centrifuge tube , pour off the supernatant carefully, and keep the sediment at the bottom of the centrifuge tube. Add 5ml of PBS, put it in a refrigerator at 4°C, and dissolve for 24 hours. The next day, in another 13ml ultracentrifuge tube, first carefully add 1ml of 60% sucrose solution, then sequentially add 1ml of 30% and 3ml of 20% sucrose solution, and finally place 5ml of the dissolved sample solution on it . After accurate weighing and balance, seal the tube and put it into an ultracentrifuge. Centrifuge at 100,000 rpm for 1 hour at 4°C. The centrifuge tube was taken out,...

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Abstract

The invention provides a hybrid virus-like particle. The particle comprises a matrix protein M1 of influenza virus, surface antigen hemagglutinin (HA) of the influenza virus and fused membrane protein, wherein the fused membrane protein comprises an extracellular domain of major epitope of infectious bronchitis virus S protein, and a transmembrane domain and an intracellular domain of the HA of the influenza virus; the extracellular domain of the main surface antigen of the infectious bronchitis virus S protein replaces the extracellular domain with approximately same length of the 5'-terminal of the HA of the influenza virus; and the surface antigen HA of the influenza virus and the surface antigen S protein of the infectious bronchitis virus are expressed simultaneously on the surface of the hybrid virus-like particle. The invention also provides a bivalent vaccine of avian influenza and infectious bronchitis, and the bivalent vaccine comprises the hybrid virus-like particle and adjuvants. The invention also provides a preparation method of the hybrid virus-like particle.

Description

technical field [0001] The invention relates to avian influenza and infectious bronchitis mixed virus-like particles, and its preparation and application. Background technique [0002] Avian influenza (Avian Influenza, AI) is an infection and / or disease syndrome of poultry (poultry and wild fowl) caused by influenza A virus of the family Orthomyxoviridae, which occurs in chickens, ducks, geese, pigeons and other birds and birds. Humans, but mainly against turkeys and chickens, mammals such as pigs, horses, seals, humans, etc. can also be infected. According to the pathogenicity of avian influenza virus (Avian Influenza Virus, AIV) strains, the type of poultry, the environment, feeding and management conditions, and concurrent diseases, the infected poultry showed asymptomatic infection, mild upper respiratory symptoms, Egg production drops to acute pathogenic death and other forms, which directly affect the health of the chicken body and product quality. It is one of the im...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/866A61K39/295A61P31/16A61P31/14A61P11/00A61K39/145A61K39/215
Inventor 曹永长刘大才薛春宜
Owner SUN YAT SEN UNIV
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