Application of rainbow trout IFN-gamma2 (Interferon-gamma2) in preparing anti-IHN (Infectious Haematopoietic Necrosis) virus product

A virus and product technology, applied in the application field of rainbow trout IFN-γ2 in the preparation of anti-IHN virus products, can solve the problems of recessive infection, high mortality of fry or juvenile fish, etc.

Active Publication Date: 2014-09-17
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Infectious Hematopoietic Necrosis (IHN) often causes high mortality in fry or juveniles, and can be insidiously infected. It is one of the threats to the rainbow trout farming industry in my country. Sensitivity of Infectious Hematopoietic Necrosis Virus (IHNV) to Interferon in Rainbow Trout

Method used

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  • Application of rainbow trout IFN-gamma2 (Interferon-gamma2) in preparing anti-IHN (Infectious Haematopoietic Necrosis) virus product
  • Application of rainbow trout IFN-gamma2 (Interferon-gamma2) in preparing anti-IHN (Infectious Haematopoietic Necrosis) virus product
  • Application of rainbow trout IFN-gamma2 (Interferon-gamma2) in preparing anti-IHN (Infectious Haematopoietic Necrosis) virus product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Obtaining of rainbow trout IFN-γ2 gene and its recombinant vector for expression

[0027] 1. Cloning of the target gene

[0028] The rainbow trout (Oncorhynchus mykiss, kept and bred in the laboratory, a conventional rainbow trout species) was killed, and the head kidney and spleen were quickly aseptically stripped on ice, and an appropriate amount of MEM medium containing 10% FBS was added. Filtered through a 70 μm nylon mesh (product of Fisher Scientific), stimulated by 5 μg / mL phytohemagglutinin, and extracted total RNA using the RNA extraction kit of Promega Company, according to the instructions of M-MLV reverse transcriptase, with primer Oligo( dT)18 was reverse transcribed to obtain the first strand of cDNA.

[0029] Referring to the published rainbow trout IFN-γ2 sequence (Genbank: FM864345.1), design upstream and downstream primers for amplifying rtIFN-γ2, upstream primer: 5'-ATC GGATCC GCTCAGTACACATCAATTAAC-3' (underlined BamHI restriction site),...

Embodiment 2

[0040] Embodiment 2, application of rainbow trout IFN-γ2 gene

[0041] 1. Protein expression and expression pattern analysis

[0042]The recombinant strain DH5α / pET32a-rtIFN-γ2 was cultured overnight in 5 mL LB medium containing 100 μg / mL AMP at 37° C. with shaking at 220 rpm. The next day, the overnight culture was inoculated in 100 mL LB medium at a ratio of 1:100, cultured with shaking at 220 rpm until the OD value reached 0.6-0.8, and the expression was induced by adding 1 mM IPTG. After 4 hours, the cells were collected by centrifugation at 4000 rpm at 4 °C.

[0043] The bacteria were resuspended in PBS with 1 / 10 of the initial medium volume, and divided into supernatant and precipitate after ultrasonic lysis and crushing. SDS-PAGE was used to analyze the existing form of the target protein.

[0044] The result is as figure 2 As shown, M is the protein molecular mass standard; 1 is before the induction of the expression strain; 2 is after the induction of the expressio...

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Abstract

The invention discloses an application of rainbow trout IFN-gamma2 (Interferon-gamma2) in preparing an anti-IHNV (Infectious Haematopoietic Necrosis Virus) product. The invention provides an application of rtIFN-gamma2 protein, an encoding gene or a recombinant carrier with the encoding gene in preparing a product for inhibiting infectious hemopoietic organ necrosis viruses. The amino acid sequence of the rtIFN-gamma2 protein is a sequence 2 as shown in a sequence table. Experiment shows that IFN-gamma2 has the anti-IHNV activity, the situation that no effective anti-IHNV preparation is available in China at present is changed, meanwhile the interferon-gamma also has the immunity adjustment activity, and the rtIFN-gamma2 can be further developed into a natural immunity enhancing agent or an adjuvant for rainbow trout.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of rainbow trout IFN-γ2 in the preparation of anti-IHN virus products. Background technique [0002] In recent years, the research enthusiasm of fish interferon has been increasing year by year, and the antiviral effect of interferon has always been the focus of this research. Earlier, in 2003, through the plaque reduction experiment, the artificially prepared Atlantic salmon type I interferon α1 subtype was confirmed to have the effect of resisting infectious pancreatic necrosis virus infection. With the deepening of research, zebrafish, rainbow trout, seven-banded grouper, and grass carp have been successively confirmed to have the ability to resist virus replication. Among them, it is worth noting that the prepared seven-banded grouper and grass carp injected with interferon can make them resistant to virus infection, which provides strong evidence for the effecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/21A61K48/00C12N15/70C12N1/21A61P31/14C12R1/19
Inventor 曹永生徐黎明赵景壮卢彤岩
Owner HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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