Fish interferon gene and use thereof

A technology of interferon and fish, applied in the field of genetic engineering, can solve the problem that the research and application of fish interferon have not made great progress

Inactive Publication Date: 2007-10-17
INST OF AQUATIC LIFE ACAD SINICA
View PDF0 Cites 16 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the existence of fish interferon has been found in many fishes since then, the solution to the problem of fish viral diseases has once shown a bright prospect (Eaton, 1990), but unfortunately, when the research on human interferon started from the experiment The research and application of fish interferon has not made great progress when the basic exploration of the laboratory is moving towards clinical application.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Fish interferon gene and use thereof
  • Fish interferon gene and use thereof
  • Fish interferon gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Virus inactivation and induction of CAB interferon in crucian carp blastula cells

[0035] Crucian carp blastula cell line (CAB) and grass carp kidney cell line (CIK) were conventionally cultured at 28°C in 199 medium (GIBCO) with 10% newborn bovine serum (inactivated at 56°C for 30 min before use). GCHV was amplified on CIK cells and virus titers were detected. GCHV was purified by differential centrifugation before inactivation by UV light: 4×10 3 g centrifugation for 20 min to remove cell residues, and the supernatant was subjected to 1×10 5 g ultracentrifugation for 1.5h to precipitate the virus, and the virus pellet was diluted with an appropriate amount of serum-free 199 medium, and continued to 1.5×10 3 g centrifuged for 20min to get the supernatant to obtain purified virus. The inactivation device uses a 30W ordinary ultraviolet germicidal lamp, fixed on the ultra-clean workbench, and the vertical distance from the shaker is 16cm. Before inactivat...

Embodiment 2

[0037] Example 2: Total RNA extraction and mRNA purification of virus-infected fish cells

[0038] Total RNA was extracted from CAB cells induced by inactivated GCHV and control CAB cells without virus induction under the same conditions by ultracentrifugation with CsTFA (cesium trifluoroacetate, Pharmacia). After the cells were induced by the virus, they were washed 3 times with PBS. Then, 4 mL of extraction buffer was added to each bottle of cells to disrupt the cells, and the cells were centrifuged at 5000 g at 15°C for 20 min to remove cell residues. The supernatant was aspirated 10 times with a No. 7 needle, and then ultracentrifuged. During ultracentrifugation, add 6.65mL CsTFA to 6 14×89mm centrifuge tubes (3 each for experimental cells and 3 control cells), add about 3.5mL of extract sample to each tube, 125000g, 15°C, use SW41 The head was ultracentrifuged for 16h. After centrifugation, add 100 μL of TE (10 mM Tris-Cl, pH 7.51 mM EDTA) to each centrifuge tube to diss...

Embodiment 3

[0040] Example 3: Synthesis of fish cell SMART cDNA infected with virus

[0041] Carry out according to the experimental procedures established in the manual of Clontech SMART PCR Synthesis Kit. The steps are: 1 μg PolyA + RNA was mixed with cDNA synthesis primer (CDS primer) and 1 μL of SMART II oligonucleotide primer, and water was added to a final volume of 5 μL. The PCR instrument was placed on ice at 72°C for 2 minutes, and then 2 μL of 5× first-strand reaction buffer was added. , DTT 1μL, 50×dNTP 1μL, PowerScript reverse transcriptase 1μL, and incubated at 42°C for 1h to synthesize the first-strand cDNA. Then take 2 μL in 100 μl system for second strand synthesis by LD-PCR. The PCR reaction parameters were: pre-denaturation at 95°C for 1min, followed by 13 cycles of 95°C for 5s, 65°C for 5s, and 68°C for 6min. The PCR product can be used for RACE-PCR to amplify the full-length cDNA of the specifically expressed gene. This experiment successfully obtained the full-len...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
body lengthaaaaaaaaaa
weightaaaaaaaaaa
body lengthaaaaaaaaaa
Login to view more

Abstract

The present invention discloses a fish interferon gene and its uses. A gene represented by SEQ ID NO:1, a protein represented by SEQ ID NO:2. The gene can express obviously in virus-infected cell and stably transplanted interferon gene cell strain. The gene expression product has anti-virus infection function in cultured cell and fish body. The crucian interferon gene can be utilized and developed in prokaryotic, eukaryotic expression system (fish cell expression system and yeast cell expression system). The product can also be used as anti-virus biological agent and pharmaceutical feedstuff additive which is convenient for use and has obvious and take effect quickly.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to a nucleotide sequence and an amino acid sequence of crucian carp interferon (Interferon, IFN), and at the same time relates to the application of the gene expression product in the antiviral effect of cultured cells, and can also be used as Use in antiviral biological agents, fish antiviral drugs and medicinal feed additives. Background technique [0002] Among the diseases caused by pathogenic microorganisms, diseases caused by viruses, such as West Nile encephalitis (WNV) and Ebola hemorrhagic fever (EBV), which are widespread in the world, are caused by viruses. Virus (HIV), severe acute respiratory syndrome (SARS), which broke out in 2003, and avian influenza (AIV, such as H5N1 type), which became popular all over the world in 2006, are seriously threatening human health (Kawai and Akira, 2006 ). Fish farming is also facing the ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/20C07K14/555A61K36/17A61K48/00A61P31/12A23K1/16A23K1/18A23K20/147A23K50/80
Inventor 张义兵桂建芳
Owner INST OF AQUATIC LIFE ACAD SINICA
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap