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Swine influenza virus-porcine reproductive and respiratory syndrome virus mixed virus-like particle, and its preparation method and use

A technology for swine blue-ear disease virus and swine influenza virus, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, and applications, can solve the problems of long production cycle, leakage and spread of live virus, poor immune effect, etc., and achieve sustainable development. Long time, good immune effect, good immune response 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 level 3 laboratory according to regulations, and the inactivation of the virus must be guaranteed to be complete and thorough
[0007] 3. Inability to distinguish vaccinated pigs from virus-infected pigs
At present, the main commercial PRRS vaccines are inactivated vaccines and attenuated vaccines. Although they can alleviate clinical symptoms after use, they cannot prevent the occurrence of virulent infection, shedding of sick pigs and persistent infection.
However, attenuated vaccines and inactivated PRRS commercial routine vaccines currently used cannot provide ideal immune protection due to potential safety hazards or poor immune effects.
What is particularly dangerous is that PRRSV has the characteristics of antibody-dependent enhancement, that is, in the presence of antibodies in pigs, PRRSV infection can increase the morbidity and mortality of pigs and cause greater losses

Method used

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  • Swine influenza virus-porcine reproductive and respiratory syndrome virus mixed virus-like particle, and its preparation method and use
  • Swine influenza virus-porcine reproductive and respiratory syndrome virus mixed virus-like particle, and its preparation method and use
  • Swine influenza virus-porcine reproductive and respiratory syndrome virus mixed virus-like particle, and its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0057] Example 2: Construction of insect baculovirus expression plasmids expressing M1, NP, HA, NA, HG genes and synthesis of recombinant insect baculoviruses in insect cells

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

[0059]Insect baculovirus plasmid PFastBac-dual (product of Invitrogen Company) was digested with restriction endonuclease Sal I / Hind III at 37°C for 3 hours, and the digested plasmid PFastBac-dual was recovered and purified with a gel extraction 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, 1ul of product recovered from enzyme digestion of PFastBac-dual plasmid, 1ul of T4 DNA ligase, and 10ul of ddH20. Use the heat shock method to transfer the ligation product into Top10 competent cells, add it to a small plastic centrifuge tube, mix gent...

Embodiment 3

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

[0076] 200ml of sf-9 cell mixture was suspended and cultured in a 1-liter Erlenmeyer shaker flask, the cell culture medium was serum-free sf-900II (or Grace insect medium from Invitrigen), the shaking speed of the shaker was 100rpm, and the temperature was constant at 27°C. When the cell concentration reaches 2×10 6 Cells / ml, sf9 cells were co-transfected with Bac-M1, Bac-NP, Bac-HA-NA, Bac-HG-NA insect baculovirus. The MOI ratio of the virus was 3 (Bac-M1 and Bac-NP): 1 (Bac-HA-NA): 1 (Bac-HG-NA). After the transfected cells were shaken and cultured at constant temperature for 3 days, all the 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 centrifugal speed of 10,000 rpm. Keep the s...

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 put 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, and tw...

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Abstract

The invention provides a swine influenza virus-porcine reproductive and respiratory syndrome virus mixed virus-like particle. The swine influenza virus-porcine reproductive and respiratory syndrome virus mixed virus-like particle contains a swine influenza virus stromatin M1, a swine influenza virus surface antigen hemagglutinin HA and an envelope fusion protein, wherein the envelope fusion protein contains an extramembranous domain of a main epitope of a porcine reproductive and respiratory syndrome virus protein GP5, and a transmembrane domain and an intramembranous domain of an influenza virus hemagglutinin HA; the extramembranous domain of the main epitope of the porcine reproductive and respiratory syndrome virus protein GP5 replaces an extramembranous domain of an end 5' of the swine influenza virus surface antigen hemagglutinin HA, and the length of the extramembranous domain of the main epitope of the porcine reproductive and respiratory syndrome virus protein GP5 is approximately equal to that of the replaced extramembranous domain; and the swine influenza virus surface antigen hemagglutinin HA and the porcine reproductive and respiratory syndrome virus protein GP5 are expressed on the surface of the swine influenza virus-porcine reproductive and respiratory syndrome virus mixed virus-like particle simultaneously. The invention also provides a bivalent vaccine for preventing swine influenza and blue ear disease. The bivalent vaccine contains the swine influenza virus-porcine reproductive and respiratory syndrome virus mixed virus-like particles and adjuvants. The invention also provides a preparation method of the swine influenza virus-porcine reproductive and respiratory syndrome virus mixed virus-like particle.

Description

technical field [0001] The invention relates to a mixed virus-like particle of swine influenza virus and porcine PRRS virus, and 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 influenza viruses can also be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C12N15/866A61K39/295A61P31/16A61P31/14A61K39/145A61K39/12
Inventor 曹永长刘大才薛春宜
Owner SUN YAT SEN UNIV
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