Mixed virus-like particle (VLP) of avian influenza and Newcastle disease, preparation method thereof and application thereof

A technology for Newcastle disease virus and influenza virus, which is applied in the field of avian influenza and Newcastle disease mixed virus-like particles, can solve the problems of long production cycle, leakage and spread of live virus, and high production conditions, and achieves long duration, good immune effect, and good immunity. effect of immune response

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 unique 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, and even An "ineffective vaccine" is produced, which is 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 product

Method used

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  • Mixed virus-like particle (VLP) of avian influenza and Newcastle disease, preparation method thereof and application thereof
  • Mixed virus-like particle (VLP) of avian influenza and Newcastle disease, preparation method thereof and application thereof
  • Mixed virus-like particle (VLP) of avian influenza and Newcastle disease, preparation method thereof and application thereof

Examples

Experimental program
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Example Embodiment

[0055] Example 2: Construction of insect baculovirus expression plasmids expressing M1, NP, HA, NA, F / HA genes and in insect cells Insect baculovirus

[0056] (1) Construction of recombinant plasmids expressing M1 and NP genes

[0057] Insect baculovirus plasmid PFastBac (product of Invitrogen) was digested with restriction enzymes Sal I / Hind III at 37°C for 3 hours, and then purified with a gel recovery kit to recover and purify the digested plasmid PFastBac. Under the action of T4 DNA ligase, the digested plasmid and 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 fragments recovered by digestion with M1, 1ul of product recovered by digestion of PFastBac plasmid, 1ul of T4 DNA ligase, and ddH2O to make up to 10ul. 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 the tube on ice for 30 minutes, tr...

Example Embodiment

[0073] Example 3: Expression of M1, NP, HA, F / HA and NA genes in co-transfected suspension cultured insect cells sf-9

[0074] Suspension culture of 200ml sf-9 cell mixture in a 1 liter triangular shaker flask, the cell culture medium is serum-free sf-900II (or Grace insect medium from Invitrigen), the shaking speed of the shaker is 100rpm, the temperature Constant at 27°C. When the cell concentration reached 2×106 cells / ml, sf9 cells were co-transfected with Bac-M1, Bac-NP, Bac-HA-NA, Bac-F / HA-NA insect baculovirus. The MOI ratio of the virus is 3 (Bac-M1 and Bac-NP): 1 (Bac-HA-NA): 1 (Bac-F / HA-NA). After the transfected cells were cultured under constant temperature shaking for 3 days, all samples were collected, centrifuged at 4°C for 30 minutes at a speed of 3000 rpm, and the supernatant was collected. After the centrifuged cell pellet was treated with cell lysate, it was centrifuged at 4°C for 10 minutes at 10,000 rpm. Save the supernatant after centrifugation. During t...

Example Embodiment

[0075] Example 4: Purification of virus-like particles and indirect immunofluorescence detection and electron microscope observation.

[0076] Put the cell supernatant collected by the above centrifugation into a 13ml ultracentrifuge tube, weigh, equilibrate, and seal the tube, put it in an ultracentrifuge (product of Bechmem), centrifuge at 4°C, 100,000rpm for 1 hour, and then take out the centrifuge tube , Pour out the supernatant carefully and save the deep 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, carefully add 1ml 60% sucrose solution, then 1ml 30% and 3ml 20% sucrose solution, and finally put 5ml of the dissolved sample solution on it . After accurate weighing and balance, seal the tube on the ultracentrifuge. Centrifuge at 100,000 rpm at 4°C for 1 hour. Take out the centrifuge tube, and collect the two bands located at the junction of 20...

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Abstract

The invention provides a mixed VLP. The mixed VLP comprises: a stromatin M1 of an influenza virus; a hemagglutinin HA of a surface antigen of the influenza virus; a nexus protein which comprises an extracellular domain of a main antigen epitope of a Newcastle disease virus (NDV) F protein, a transmembrane domain and an intracellular domain of the hemagglutinin HA of the influenza virus, wherein the extracellular domain of the main antigen epitope of the NDV-F protein replaces an extracellular domain which is at 5'-terminus of the hemagglutinin HA of the influenza virus and has a roughly same length; and the hemagglutinin HA of the influenza virus and the surface antigen F protein of the NDV which are simultaneously expressed on the surface of the mixed VLP. The invention also provides a divalent vaccine of avian influenza and the NDV and a method for preparing the mixed VLP, wherein the divalent vaccine contains the mixed VLP and an adjuvant.

Description

technical field [0001] The invention relates to avian influenza and Newcastle disease 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 import...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/866A61K39/295A61P31/16A61P31/14A61K39/145A61K39/17
Inventor 曹永长刘大才薛春宜
Owner SUN YAT SEN UNIV
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