Grass carp hemorrhage vaccine prepared through yeast display and preparation method of grass carp hemorrhage vaccine

A grass carp hemorrhagic disease and vaccine technology, applied in the field of genetic engineering, can solve problems such as affecting the efficiency of yeast display vaccine, and achieve the effects of cheap production and simple cultivation

Inactive Publication Date: 2016-08-24
INST OF AQUATIC LIFE ACAD SINICA +1
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The N-linked sugar chain is composed of up to 200 mannose residues, thereby forming hyperglycosylation (Dean,. Asparagine-linked glycosylatio

Method used

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  • Grass carp hemorrhage vaccine prepared through yeast display and preparation method of grass carp hemorrhage vaccine
  • Grass carp hemorrhage vaccine prepared through yeast display and preparation method of grass carp hemorrhage vaccine

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Embodiment Construction

[0022] 1. Use glass beads to crush the yeast cell wall of Saccharomyces cerevisiae BY4741△Mnn1 knocked out of the Mnn1 gene, and use phenol and chloroform to extract genomic DNA from the yeast cell lysate. The specific operation process is as follows:

[0023] 1.1 Inoculate Saccharomyces cerevisiae cells BY4741△Mnn1 into 1ml YPD (containing 2% glucose) medium, culture at 30°C and 200 rpm for 16 hours with shaking;

[0024] 1.2 Take 1.5ml of yeast liquid, centrifuge at 6000 rpm for 2 minutes, and take the precipitate;

[0025] 1.3 Add 200 μl extraction solution to resuspend the cells;

[0026] 1.4 Add 0.3g glass beads, 200μl each of phenol and chloroform, and vortex for 3 minutes;

[0027] 1. Centrifuge at 512000 rpm for 10 minutes, take the supernatant and add it to a new centrifuge tube;

[0028] 1.6 Add 2 times the volume of pre-cooled absolute ethanol, mix the solution thoroughly, and put it in a -20°C refrigerator for 30 minutes;

[0029] 1. Centrifuge at 12000 rpm for ...

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Abstract

The invention discloses a grass carp hemorrhage vaccine prepared by adopting a yeast surface display technology, relating to the preparation of genetic engineering vaccines. The grass carp hemorrhage vaccine is prepared in a manner that GCRV-VP7 protein is displayed on the surface of brewer's yeast cells EBY100 delta Mnn9 with glycosylated genes knocked out. The brewer's yeast is an expression system of eukaryon, the galactosylated modification is carried out on the virus coat protein expressed by the brewer's yeast, and then an immune system of the grass carp is induced to generate virus-neutralizing antibodies. The grass carp hemorrhage vaccine has the advantages that a used yeast strain EBY100 delta Mnn9 with glycosylation deficiency can effectively remove the super glycosylation of the protein on the surface of the yeast, and thus the problem that the GCRV-VP7 antigen is covered, consequently, the vaccine efficiency loss and decrease are caused, is solved; the yeast cells are easy to culture and low in preparation cost, and thus the grass carp hemorrhage vaccines can be prepared in large scale at low cost.

Description

technical field [0001] The invention relates to genetic engineering technology, more specifically to the preparation of genetic engineering vaccines. Background technique [0002] Grass carp reovirus (GCRV) is a double-stranded RNA virus belonging to the genus Aquareoviridae, and is currently the most toxic aquatic reovirus (Rangelet al, Identification of grass carp haemorrhage virus as a new genogroup of aquareovirus, J Gen Virol, 1999(80):2399-2402). The virus caused hemorrhagic disease in grass carp, the main freshwater aquaculture species in my country, and the mortality rate was as high as 90%, which caused huge losses to my country's aquaculture industry (Liu et al., Prokaryotic expression, purification and immunization of grass carp hemorrhagic disease virus VP6 protein Effect, Acta Aquatica Sinica, 2012(03):429-435). In addition to grass carp, GCRV can also infect black carp (Mylopharyngodon piceus), wheat ear carp (Pseudorasbora parva) and rare gobiocypris rarus (D...

Claims

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Application Information

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IPC IPC(8): A61K39/15A61K39/00A61P31/14C12N1/19C12N15/46C12R1/865
Inventor 袁丽方勤罗绍祥闫利明戴和平张晓华
Owner INST OF AQUATIC LIFE ACAD SINICA
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