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Broad-spectrum safe anti influenza A virus vaccine for animals

A type of influenza A virus and vaccine technology, applied in the field of genetic engineering, can solve the problems of long production cycle, leakage and spread of live virus, and high production conditions

Active Publication Date: 2010-02-10
许雁
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] 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
[0010] 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 strictly controlled conditions according to regulations, and the inactivation of the virus must be guaranteed to be complete and thorough
[0011] 3. Inability to distinguish vaccinated birds from those infected with real virus

Method used

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  • Broad-spectrum safe anti influenza A virus vaccine for animals
  • Broad-spectrum safe anti influenza A virus vaccine for animals
  • Broad-spectrum safe anti influenza A virus vaccine for animals

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0128] Example 2: Construction of insect baculovirus expression plasmids expressing M1, HA, NA and 3xM2eNP genes and synthesis of recombinant insect baculoviruses in insect cells.

[0129] (1) Construction of a recombinant plasmid expressing the M1 gene: Insect baculovirus expression plasmid pFastBac-Dual (product of Invitrogen Company) was digested and hydrolyzed by restriction endonuclease BamHI / NotI overnight at 37°C, then gel electrophoresis and column extraction purification. Under the action of T4 DNA ligase, the digested plasmid and the digested M1 gene DNA fragment were ligated overnight at 16°C. The reaction system is as follows: 1ul of 10×T4 ligation buffer, 3ul of the DNA fragment recovered by M1 digestion, 1ul of the recovered product of pFastBac-Dual plasmid, 1ul of T4 DNA ligase, and 10ul of ddH2O. The ligation product was transformed into E.coli DH5a competent bacteria by heat shock method. The specific operation is as follows: transfer 50ul DH 5a competent ce...

Embodiment 3

[0138] Example 3: Expression and analysis of M1, HA, NA and fusion protein 3xM2eNP genes in co-transfected suspension cultured insect cells Sf-21.

[0139] (1) Gel protein electrophoresis to detect the expression of the M1 gene: the cell centrifugation precipitate used to amplify the culture of the recombinant baculovirus Bac-M1 was treated with the cell lysis buffer, centrifuged at 4°C (13,000rpm) for 10 minutes, and collected The supernatant was subjected to gel electrophoresis to analyze whether the matrix protein M1 was expressed in the insect cell Sf-21. Briefly, 10ul of 2XSDS loading buffer was added to 10ul of cell lysate supernatant, treated at 100°C for 5min, and then centrifuged rapidly for 5 seconds. All 20ul of mixed samples were added to 4%-12% SDS-polyacrylamide gel (Invitrogen company product) spotting wells. Set the electrophoresis conditions as a constant voltage of 100V, a temperature of 4°C, and a time of 2 hours. The electrophoresis buffer was Tris-glycin...

Embodiment 4

[0142] Example 4: The intervention of nucleoprotein NP can increase the yield of virus-like particles.

[0143] As mentioned above, nucleoprotein NP can couple with matrix protein M1, and can bring the M1 protein that re-enters the host cell nucleus back to the cytoplasm [Document 14]. The synthesis amount of M1 protein directly determines the formation amount of influenza A virus granules [References 5, 6]. Recombinant insect baculovirus Bac-M1 and Bac-HANA with known virus titers were co-transfected into insect cell Sf-21. At the same time, the recombinant insect baculoviruses Bac-M1, Bac-HANA and Bac-3xM2eNP with known virus titers were co-transfected into insect cells Sf-21 in another shake flask. After culturing at 27°C for three days, the cell supernatants in the two shake flasks were collected, and the HA titer of the virus-like particles was detected by microplate counting method. The specific operation is as follows: PBS is added to each well of a 96-well cell cultu...

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Abstract

Recombinant virus-like particle contains influenza A virus matrix protein M1, surface film proteins HA and NA and M2eNP fusion protein or protein obtained by modification of mutant of at least one protein and the rest proteins, and the recombinant virus-like particle is non-replicative; wherein the M2eNP fusion protein is polymer formed by one M2e polypeptide or a plurality of M2e polypeptides atthe external end of cell membrane of matrix protein M2 or is formed by fusion of nucleoprotein NP and polymer formed by one M2e polypeptide through artificial modification or a plurality of M2e polypeptides after modification; and the nucleoprotein NP is coupled with recombinant matrix protein M1 after recombination expression and embedded in the recombinant virus-like particle. The vaccine produced by the recombinant virus-like particle can be directly applied to various animals for prevention of infection and spread of influenza A virus. The vaccine is safe in use and obvious in immune effect. Production period is short, technical operation is simple, and no purification is required, thus the vaccine is low in cost.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to recombinant virus-like particles using the insect baculovirus-insect cell expression system and an anti-influenza A virus vaccine for animals containing the recombinant virus-like particles. Background technique [0002] Influenza A viruses can be transmitted among humans, mammals and birds. For example, avian influenza is a widespread infectious disease in poultry including farmed poultry. This infectious disease virus, especially the highly pathogenic subtype virus H5N1, poses a great threat to birds. The characteristics of its symptoms are: sudden outbreak of severe disease, and rapid spread, and the death rate can be within 48 hours. According to reports, all outbreaks of highly pathogenic avian influenza are caused by H5 and H7 subtype viruses. In the past 10 years, the records of large-scale poultry bird deaths caused by this highly pathogenic avian influenza virus have c...

Claims

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Application Information

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IPC IPC(8): C12N7/01A61K39/145A61P31/16C12R1/93
Inventor 许雁曹永长李卓诺曼.吉利卡
Owner 许雁
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