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A kind of grass carp reovirus fibril-like spike vp56 protein subunit vaccine and its preparation method and application

A reovirus and subunit vaccine technology, applied in the fields of genetic engineering and molecular immunology, can solve the problems of preparing subunit vaccines, grass carp reovirus-like spike-like VP56 protein, etc., and achieve good immunogenicity , the effect of simple preparation method

Active Publication Date: 2019-11-05
HENAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report on the use of grass carp reovirus fibrillar spike-like VP56 protein to prepare subunit vaccines

Method used

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  • A kind of grass carp reovirus fibril-like spike vp56 protein subunit vaccine and its preparation method and application
  • A kind of grass carp reovirus fibril-like spike vp56 protein subunit vaccine and its preparation method and application
  • A kind of grass carp reovirus fibril-like spike vp56 protein subunit vaccine and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Preparation of grass carp reovirus fibrillar VP56 protein subunit vaccine

[0030] 1. Construction of plasmid pET32a-VP56

[0031] Grass carp reovirus was isolated from a breeding pond in Xinxiang City, Henan Province. Grass carp viscera suffering from grass carp hemorrhagic disease were obtained, mixed with PBS, ground and homogenized, and the homogenate was centrifuged at 8000g, and the supernatant was filtered through a filter with a pore size of 0.22µm. Inoculated into the grass carp kidney cells (CIK) cultured in vitro, the cells were collected after one week of culture, and the cells contained the proliferating grass carp reovirus. RT-PCR detection showed that the grass carp reovirus was type II grass carp reovirus. Using the grass carp reovirus genomic RNA as a template, primers S7F1: 5'-TTCGGTACCAGTGGAAAATTAGACA-3' and S7R1: 5'-GCACTCGAGGTACTTACAGCAAACTACC-3' were used for reverse transcription PCR. 1µL each, 94°C for 5min, immediately put on ice, then RNasin ...

Embodiment 2

[0035] Application of grass carp reovirus fibrillar VP56 protein subunit vaccine

[0036] 1. Subunit vaccines can significantly increase the number of immune cells in fish

[0037] 60 healthy grass carp fry (about 20 g per fish) were randomly divided into two groups, 30 fish in each group, as the experimental group and the control group, respectively. Inject an equal volume of PBS and raise at 25°C. On the 1st, 3rd, 5th, 7th, 14th, 21st, 28th, and 35th days after injection, 3 fish were taken from each of the two groups every day, and the blood was taken from the tail vein, and diluted with a volume ratio of 1:200 using a Neubauer counting board The number of immune cells was counted and observed with Giemsa staining, and the proportions of monocytes, neutrophils and lymphocytes among one hundred immune cells were counted. The results showed that the number of immune cells in the experimental group increased significantly on the 5th day of immunization, and reached the peak v...

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Abstract

The invention discloses a grass carp reovirus spike VP56 protein subunit vaccine, as well as a preparation method and application thereof, and belongs to the technical fields of genetic engineering and molecular immunology. According to the technical scheme, the grass carp reovirus spike VP56 protein subunit vaccine is characterized in that a subunit vaccine protein is coded by a grass carp reovirus S7 gene segment, a base sequence of the grass carp reovirus S7 gene segment is shown as SEQ ID No. 1 in a sequence table, the coded protein is a grass carp reovirus spike VP56 protein subunit vaccine protein, and an amino acid sequence of the subunit vaccine protein is shown as SEQ ID No. 4 in the sequence table. After the subunit vaccine protein is injected into grass carp bodies, immune cellproliferation, antiviral related gene up-regulated expression and specific antibody proliferation of the grass carp bodies can be remarkably stimulated, the capability of grass carps in resisting grass carp reovirus infection is effectively improved, and the grass carp reovirus spike VP56 protein subunit vaccine is used for preventing grass carp hemorrhage caused by viruses in aquaculture.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and molecular immunology, and in particular relates to a grass carp reovirus-like fibrillary spike VP56 protein subunit vaccine and its preparation method and application. Background technique [0002] Grass carp reovirus (GCRV) mainly infects grass carp fry within one year old and causes grass carp hemorrhagic disease. The main symptoms are blackening of the body surface and the end of fin rays, enlarged abdomen, exophthalmos, mandible and fin base congestion , There was obvious bleeding in the mouth, muscles, and internal organs. The onset season of grass carp hemorrhagic disease is long, and the main epidemic season is from late June to the end of September every year. The peak season is August and September, and it is most popular when the water temperature is 25-30°C. The main diseases of grass carp farming in my country include grass carp hemorrhagic disease, enteritis disease, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/15A61P31/14C12N15/70C12N15/46C12P21/02
CPCA61K39/12A61K2039/53A61K2039/552C07K14/005C12N15/70C12N2720/12022C12N2720/12034
Inventor 裴超高岩孔祥会孙效迎
Owner HENAN NORMAL UNIV
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