Grass carp hemorrhage antigenic protein, preparation method and application thereof

A technology of grass carp hemorrhagic disease and antigenic protein, which is applied in the direction of virus antigenic components, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as difficulty in widespread application, high cost, complicated preparation process, etc., and achieve quality Control, method is simple, cultivate simple effect

Inactive Publication Date: 2012-10-03
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex preparation process and high cost of subunit vaccines, it is difficult to widely popularize and apply them.

Method used

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  • Grass carp hemorrhage antigenic protein, preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1: Isolation of grass carp reovirus GCRV-104

[0056] Grass carp samples suspected of suffering from grass carp hemorrhagic disease were collected in Wencun, Jiangling County, Jingzhou City, Hubei Province. The liver, spleen, kidney and other visceral tissues of the diseased fish were collected in a sterile petri dish, cut into pieces with sterile ophthalmic scissors, and added 10 Double the volume (V / W) of PBS, together with the tissue fragments, were transferred to a glass homogenizer and ground into a tissue homogenate under the condition of an ice bath. The tissue homogenate was frozen at -80°C and thawed at room temperature, and the freezing and thawing was repeated three times. Centrifuge at 5000×g for 30 min at 4°C, and filter the supernatant with a 0.22 μm filter to sterilize. Take 1mL of tissue homogenate and inoculate 10μg of co-infection agent Polybrene into CIK cells grown to a confluent monolayer, and place them in a 28°C incubator for 1h, durin...

Embodiment 2

[0057] Example 2: Extraction of total RNA of grass carp reovirus

[0058] Grass carp reovirus 104 strain is the standard strain preserved in my country: Grass carp reovirus GCRV-104 preservation number CCTCC NO: V201217.

[0059] Cultivate grass carp kidney cells to a confluent monolayer, suck out the culture medium, and inoculate 1 mL of grass carp reovirus cytotoxic material that has been centrifuged at 4000 r / min for 5 min to remove cell debris at a dose of 0.1 multiplicity of infection, and add 10 μL of Polybrene (final concentration 10 μg / ml), placed in a 28°C incubator for 1 hour to allow the virus to adsorb to the cells, gently shake the culture bottle once every 20 minutes during the adsorption process, so that the virus liquid can fully and evenly contact with the cell monolayer, after 1 hour of adsorption, discard the virus liquid. , add 5mL of 2% fetal bovine serum (V / V) culture medium, incubate at 28°C until the cells show obvious cytopathic effect, collect cytopa...

Embodiment 3

[0073] Example 3: Construction of recombinant plasmid pPICZ B-VP6 and transformation of yeast

[0074] 1. Cloning of the target gene:

[0075] Firstly, the aforementioned reverse transcription polymerase chain reaction product and the pPICZ B vector were digested with EcoRI and SacII respectively, and then the two products were ligated under the action of ligase to construct the pPICZ B-VP6 expression vector. It was confirmed by polymerase chain reaction identification and enzyme digestion identification. The positive positive plasmid pPICZ B-VP6 was identified as a recombinant plasmid with a complete grass carp reovirus VP6 protein gene. Sequencing results showed that the positive plasmid pPICZ B-VP6 contained the reading frame of the grass carp reovirus VP6 protein gene.

[0076] 2.1 Preparation of reagents

[0077] 10×YNB: Weigh 13.4g of YNB (YNB is amino acid-free yeast nitrogen source, a component of yeast medium) dissolved in 100ml of deionized water, heated until YNB i...

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Abstract

The invention discloses a grass carp hemorrhage antigenic protein, a preparation method and an application thereof, wherein the preparation method comprises the steps: A. according to nucleotide complete sequence of a grass carp reovlrus VP6 gene, designing a primer to perform reverse transcription-polymerase chain reaction; cloning to obtain a VP6 protein gene; B. using EcoRI and SacII to perform enzyme digestion on a product obtained from reverse transcription-polymerase chain reaction in step A and a pPICZB carrier; constructing a pPICZB-VP6 expression carrier; C. electrically transferring recombinant obtained in step B to pichia yeast; coating the pichia yeast on the YPDS flat plate containing ZEOCIN antibiotic, and screening to obtain a high-copy recombinant yeast containing a target gene; and D. obtaining the used antigenic protein by separation and purification. The method is simple and is easy to operate, can be used for carrying out large-scale industrial production by a fermentation tank; the protein is safe and reliable, has immunological competence and can be fully used as the grass carp hemorrhage antigenic protein.

Description

technical field [0001] The present invention relates to the technical field of biological protein preparation, more specifically to a grass carp hemorrhagic disease antigenic protein, and also to a preparation method of grass carp hemorrhagic disease antigenic protein, and to a grass carp hemorrhagic disease antigenic protein in the preparation of grass carp hemorrhagic disease vaccine Applications. Background technique [0002] Grass carp (Ctenopharyngodon idellus) is one of the most important freshwater aquaculture fishes in my country, and plays an important role in freshwater aquaculture in China. Grass carp has a wide source of feed, fast growth, and delicious meat, which is deeply loved by farmers and consumers. However, many diseases of grass carp and low survival rate of aquaculture have hindered the development of grass carp aquaculture. Grass carp haemorrhagic disease is a viral disease that seriously harms grass carp species. It has a wide range of prevalence, f...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/46C12N15/81C12N1/19C07K14/14A61K39/15A61P31/14C12R1/93C12R1/84
Inventor 周勇曾令兵苏岚
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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