Mixed virus-like particles of swine influenza virus and foot and mouth disease virus, preparation method and application thereof

A technology of porcine foot-and-mouth disease virus and influenza virus, applied in chemical instruments and methods, botany equipment and methods, methods based on microorganisms, etc., can solve problems such as long production cycle, high production conditions, leakage and diffusion of live viruses, and achieve immunity Strong, long-lasting, good immune effect

Active 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

[0005] 1. Long production cycle: It takes 5-8 months from the acquisition of new subtype virus strains to the production and marketing of vaccines, making it difficult to contain new outbreaks
[0006] 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

Method used

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  • Mixed virus-like particles of swine influenza virus and foot and mouth disease virus, preparation method and application thereof
  • Mixed virus-like particles of swine influenza virus and foot and mouth disease virus, preparation method and application thereof
  • Mixed virus-like particles of swine influenza virus and foot and mouth disease virus, preparation method and application thereof

Examples

Experimental program
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Example Embodiment

[0058] Example 2: Construction of insect baculovirus expression plasmids expressing M1, NP, HA, NA, HF genes and synthesis and recombination of insect baculovirus in insect cells

[0059] (1) Construction of recombinant plasmids expressing M1 and NP genes

[0060] Insect baculovirus plasmid PFastBac-dual (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-dual. 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 fragment recovered by digestion with M1, 1ul of product recovered by digestion of PFastBac-dual plasmid, 1ul of T4DNA ligase, and ddH2O to make up to 10ul. Use the heat shock method to transfer the ligation product into Top10 competent cells and add it to a small plastic centrifuge tube. After gentl...

Example Embodiment

[0076] Example 3: Expression of M1, NP, HA, HF and NA genes in co-transfected suspension cultured insect cells sf-9

[0077] 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 and the temperature is constant. At 27°C. When the cell concentration reaches 2×10 6 When cells / ml, use Bac-M1, Bac-NP, Bac-HA-NA, Bac-HF-NA insect baculovirus to co-transfect sf9 cells. The MOI ratio of the virus is 3 (Bac-M1 and Bac-NP):1 (Bac-HA-NA):1 (Bac-HF-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 th...

Example Embodiment

[0078] Example 4: Purification of virus-like particles and indirect immunofluorescence detection and electron microscope observation.

[0079] 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, first carefully add 1ml of 60% sucrose solution, then add 1ml of 30% and 3ml of 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 th...

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Abstract

The invention provides a mixed virus-like particles, containing stromatin M1 of influenza virus, surface antigen erythrohemagglutinin HA of influenza virus and a nexus protein containing main antigenic epitope contained capsid protein of foot and mouth disease virus and a transmembrane zone and an inner membrane zone of the erythrohemagglutinin HA of influenza virus. The main antigenic epitope contained capsid protein of foot and mouth disease virus substitutes a basically same length outer membrane zone of a 5' terminal of the erythrohemagglutinin HA of influenza virus. Surface antigen erythrohemagglutinin HA of influenza virus and the main antigenic epitope contained capsid protein of foot and mouth disease virus are expressed simultaneously on surfaces of the mixed virus-like particles. The invention also provides a divalent vaccine of swine influenza and foot and mouth disease; and the vaccine contains the mixed virus-like particles and adjuvants. The invention also provides a method for preparing the mixed virus-like particles.

Description

technical field [0001] The invention relates to a mixed virus-like particle of swine influenza virus and porcine foot-and-mouth disease virus, as well as its preparation and application. Background technique [0002] Swine influenza is an acute, febrile and highly contagious respiratory infectious disease caused by swine influenza virus (SIV) in pigs of different ages, sexes and breeds. Coughing, dyspnea, prostration, and death are the hallmarks. Pigs of all ages, sexes, and breeds can be infected, and the incidence rate is high. Infection with swine influenza virus alone can lead to a decline in pig production performance, increased feed-to-meat ratio, and slowed weight gain, causing certain economic losses to pig farms. What's more serious is that pigs are "mixers" for the recombination and replication of poultry, human, and swine influenza viruses. Swine influenza viruses have the ability to infect humans to the maximum without recombination. Moreover, early human infl...

Claims

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Application Information

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IPC IPC(8): A61K39/295A61K39/145A61K39/135C07K19/00C12N15/866A61P31/14A61P31/16C12R1/93
Inventor 曹永长刘大才薛春宜
Owner SUN YAT SEN UNIV
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