Grass carp reovirus S11 gene eukaryotic expression recombinant plasmid preparation method and application thereof in serving as nucleic acid vaccine

A technology of reovirus and recombinant plasmid, applied in the fields of genetic engineering and molecular immunology, to achieve good immunogenicity and simple preparation method

Inactive Publication Date: 2018-03-06
HENAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are very few types of vaccines prepared for Type II viruses, mainly subunit vaccines and nucleic acid vaccines for the VP4 protein of Type II viruses, while Type I viruses and Type II viruses have obvious differences in protein structure and composition. Virus vaccines cannot provide the same protection against type II viruses, so it is necessary to find more available antigenic sites for type II viruses and develop more types of vaccines
The VP35 protein encoded by the S11 segment of type II virus is predicted to be the main capsid protein of this virus. So far, there is no relevant report about using the protein gene as a nucleic acid vaccine. Therefore, this patent provides a method for constructing nucleic acid using this gene. Vaccine method, and verified the actual application effect of nucleic acid vaccine

Method used

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  • Grass carp reovirus S11 gene eukaryotic expression recombinant plasmid preparation method and application thereof in serving as nucleic acid vaccine
  • Grass carp reovirus S11 gene eukaryotic expression recombinant plasmid preparation method and application thereof in serving as nucleic acid vaccine
  • Grass carp reovirus S11 gene eukaryotic expression recombinant plasmid preparation method and application thereof in serving as nucleic acid vaccine

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Experimental program
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Effect test

Embodiment 1

[0030] Preparation of Grass Carp Reovirus S11 Target Gene Nucleic Acid Vaccine

[0031] 1. Construction of grass carp reovirus recombinant plasmid S11-pcDNA3.1

[0032] 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 buffer, 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 , the filtrate was inoculated into the grass carp kidney cells (CIK) cultured in vitro, and the cells were collected after one week of culture, and the cells contained the proliferating grass carp reovirus. The total RNA of cells and viruses was extracted, and RT-PCR detection showed that the grass carp reovirus was type II grass carp reovirus. Grass carp reovirus genomic RNA was used as a template, and primers S11F: 5'-TGTGGATCCACCAATTATCGGTAAGTATGGAA-3' and primers S11R: 5'-CTG...

Embodiment 2

[0038] Application of Grass Carp Reovirus Recombinant Plasmid S11-pcDNA3.1 as Nucleic Acid Vaccine

[0039] 1. Grass carp reovirus S11 target gene nucleic acid vaccine can significantly stimulate the increase in the number of immune cells in fish

[0040] 90 healthy grass carp fry (about 20 g per fish) were randomly divided into 3 groups, 30 fish in each group, as the experimental group, the empty plasmid control group and the negative control group respectively, and each fish in the experimental group was intramuscularly injected with nucleic acid vaccine 0.5 μg / g Fish body weight, the empty plasmid control group was injected with the same amount of pcDNA-3.1 empty vector plasmid, the negative control group was injected with the same volume of PBS buffer, and raised at 25°C. On the 1st, 7th, 14th, 21st, 28th, 35th, 42nd, and 49th day after injection, 3 fish were taken from each of the 3 groups every day, and the blood was taken from the tail vein, diluted with a volume ratio ...

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Abstract

The invention discloses a grass carp reovirus S11 gene eukaryotic expression recombinant plasmid preparation method and application thereof in serving as nucleic acid vaccine, and belongs to the technical field of gene engineering and molecular immunology. The preparation method disclosed by the invention is characterized by comprising the steps of extracting viral genome RNA, performing reverse transcription to convert the viral genome RNA into cDNA, amplifying a corresponding DNA sequence out, constructing the DNA sequence to pcDNA-3.1(+) plasmid, converting Escherichia coli DH5alpha, screening out positive clone bacteria containing recombinant plasmid, culturing a lot of the positive bacteria and extracting recombinant plasmid S11-pcDNA3.1 contained in the bacteria. The recombinant plasmid is utilized as nucleic acid vaccine to perform intramuscular injection on the grass carps, and the nucleic acid vaccine enters the muscle cells and expresses VP35 protein of the grass carp reovirus in the muscle cells; thus, fish body immune cell proliferation is stimulated, antiviral related gene expression is up regulated, fish bodies are stimulated to generate antiviral antibodies, and capability of grass carps in resisting grass carp reovirus infection is effectively improved; furthermore, the grass carp reovirus S11 gene eukaryotic expression recombinant plasmid can be used for preventing a grass carp hemorragic disease caused by the grass carp reovirus 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 preparation method of a grass carp reovirus S11 gene eukaryotic expression recombinant plasmid and its application as a nucleic acid vaccine. Background technique [0002] Grass carp reovirus (GCRV) infects one to two-year-old grass carp fry, causing blackening of the body surface and the end of the fin rays, swelling of the abdomen, exophthalmos, mandibular and fin base hyperemia, oral cavity, muscles, Visceral bleeding is called grass carp hemorrhagic disease. Since the grass carp reovirus was discovered and isolated in the 1980s, it has been found that the virus has a wide range of epidemics, including Guangdong, Guangxi, Lianghu, Anhui, Jiangxi, Guizhou, Henan and other places. The epidemic time is long, and it can be from June to October Epidemic, the high temperature season in summer is prone to outbreaks, which can cause ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66A61K39/15A61P31/14
CPCA61K39/12A61K2039/53A61K2039/552C12N15/66C12N15/85C12N2720/12034
Inventor 裴超高岩孔祥会孙效迎李莉吕爱军
Owner HENAN NORMAL UNIV
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