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Recombination infectious haematopoietic necrosis virus (rIHNV HLJ-09) strain and construction method and application thereof

A hematopoietic organ necrosis, HLJ-09 technology, applied in the biological field, can solve the problems of difficult directional operation and high mutation rate of RNA virus

Active Publication Date: 2015-09-16
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mutation rate of RNA viruses is high, and it is difficult to directly manipulate them

Method used

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  • Recombination infectious haematopoietic necrosis virus (rIHNV HLJ-09) strain and construction method and application thereof
  • Recombination infectious haematopoietic necrosis virus (rIHNV HLJ-09) strain and construction method and application thereof
  • Recombination infectious haematopoietic necrosis virus (rIHNV HLJ-09) strain and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Obtaining of Recombinant Infectious Hematopoietic Necrosis Virus Strain rIHNV HLJ-09

[0040] Specific steps are as follows:

[0041] 1. Construction of full-length cDNA recombinant plasmid

[0042] Use primer5.0 to analyze the restriction enzyme cutting sites of the whole gene of HLJ-09 strain virus (Genbank accession no. JX649101), select 5 single restriction enzyme cutting sites, and the full-length cDNA is blocked from 5' to 3' direction The internal and introduced enzyme cutting sites SnaB I, Nde I, Spe I, Nhe I, Mlu I, and Nco I are divided into lengths of 1696bp (F1), 1734bp (F2), 1852bp (F3), 1707bp (F4) respectively. ), 6 fragments of 2140bp (F5) and 1997bp (F6) were amplified by RT-PCR, and the two restriction sites of SnaB I and Spe I were nonsense mutations, which were used as molecular markers for the difference between the recombinant virus and the parental virus. The primers and amplified segments required for genome segment amplification are ...

Embodiment 2

[0069] Example 2 Application of rIHNV HLJ-09 strain in virus vector

[0070] Specific steps are as follows:

[0071] 1. Rescue of recombinant virus rIHNV-EGFP

[0072] Using the rIHNV HLJ-09 reverse genetics system, the NV ORF reporter gene of rIHNV HLJ-09 was replaced with the EGFP (green fluorescent protein) coding gene to construct the recombinant plasmid pIHNV-EGFP; The L15 in FBS was reared in a 24-well plate, and the cell density was about 80%-90%. Add 1 μg pIHNV-EGFP and 0.5 μg pCI-N, 0.5 μg pCI-P, 0.2 μg pCI-L, 0.1 μg pCI-NV, 0.1 μg pCI-G plasmids to 200 μL serum-free L15 medium, and mix gently uniform. Add 0.6 μL PLUSTM Reagents to the DNA mixture, mix gently, and incubate at room temperature for 5 minutes. Add 200 μL of the above transfection solution into CHSE-214 cells, and gently shake the 24-well plate back and forth. Place the transfection plate in an incubator at 25°C for 6 hours, discard the liquid, and add freshly prepared L15 culture liquid containing 1...

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Abstract

The invention relates to a recombination infectious haematopoietic necrosis virus (rIHNV HLJ-09) strain and a construction method and application thereof, and belongs to the field of biotechnologies. The microorganism preservation number of the rIHNV HLJ-09 strain is CGMCC No.8606, and the passage experiment of cells infected with recombinant virus shows that the virus can achieve passage stability. The rIHNV HLJ-09 is used in the intraperitoneal inoculation of juvenile rainbow trout, the result leads to the disease and death of the juvenile rainbow trout, and the lethallty rate can reach 90%, so that it shows that the virus has the similar pathogenic character as parental generation HLJ-09 strain virus. Furthermore, a rIHNV HLJ-09 reverse genetic system is used for replacing reporter gene green fluorescent protein (EGFP) encoding genes with NV ORF in the rIHNV HLJ-09, the virus is successfully rescued, and the rescued virus is named as rIHNV-EGFP. When a laser scanning confocal microscope is used for observing the cells infected with the rIHNV-EGFP virus, obvious green fluorescent can be observed, and it shows that the rIHNV HLJ-09 virus strain can be used for expressing foreign proteins.

Description

technical field [0001] The invention relates to a recombinant infectious hematopoietic organ necrosis virus rIHNV HLJ-09 strain and its construction method and application, belonging to the field of biotechnology. Background technique [0002] Infectious haematopoietic necrosis (IHN) is an infectious hematopoietic necrosis virus (IHNV) that infects salmonids with hemopoietic organ hemorrhage and necrosis. A highly contagious and acute viral disease with predominant pathological features. IHNV is a typical representative of fish rhabdoviruses, which harms a wide range of fish species and has the ability to infect all species of salmonidae. IHNV highly virulent strains have a lethality rate of up to 100% for juvenile salmon and trout, and the surviving fish carry the virus for life, become potential sources of infection, and excrete pathogens through feces and sperm eggs. At present, the disease is widely prevalent in North America, Europe, and Asian countries, and has becom...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/85C12N15/47C12N15/86C12R1/93
Inventor 刘敏李一经赵丽丽唐丽杰乔薪瑗
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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