Preparation and application of anti-IHNV kelp extract

A kelp extract and a technology of kelp are applied in the field of preparation and application of anti-IHNV kelp extract, and can solve the problems of no effective commercial vaccine, economic loss of rainbow trout breeding and the like

Pending Publication Date: 2022-04-26
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Under natural conditions, the virulent strain of Infectious hematopoietic necrosis virus (IHNV) can cause nearly 100% mortality of rainbow trout fry, which has brought huge economic losses to the rainbow trout breeding industry in my country.
In recent year

Method used

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  • Preparation and application of anti-IHNV kelp extract
  • Preparation and application of anti-IHNV kelp extract
  • Preparation and application of anti-IHNV kelp extract

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, the preparation of sea-tangle extract

[0053] 1.1, preparation of kelp crude extract

[0054] 1.1.1, preparation of kelp extract

[0055] The preparation of kelp extract comprises the following steps:

[0056] Step 1, pulverize the dried kelp (Laminaria japonica) (composed of fronds and stalks) through a 20-mesh sieve to obtain dry kelp powder; mix distilled water with dry kelp powder to make the liquid-material ratio reach 30:1 (that is, distilled water and kelp The mass ratio of the dry powder is 30:1), soaked for 6 hours to obtain the kelp soaking liquid.

[0057] Step 2, enzymatic hydrolysis: place the kelp soaking solution obtained in step 1 in an ultrasonic oscillator (ultrasonic power 360W) for 30 minutes, adjust the pH value of the kelp soaking solution to 5.0 with HCl or NaOH, add cellulase, 55 ° C enzyme hydrolyzed cellulose for 20 minutes; adjust the pH value of the cellulose hydrolyzate to 4.2 with HCl or NaOH; The pH value of the enzymati...

Embodiment 2

[0074] Embodiment 2, kelp extract is analyzed to cell safety

[0075] 2.1. Measurement method

[0076] EPC cells were added to each well at 1.0×10 5 Cells per 100 μL were seeded in 96-well plates and cultured for 24 hours to conduct cell safety analysis. The kelp extract was dissolved in cell maintenance solution to make the content of 100, 200, 300, 400, 500, 1000 and 2000 μg / mL, respectively. After discarding the 96-well plate culture solution (10% FBS, 100U / ml penicillin and 100U / ml streptomycin were added to the MEM medium), 100 μL of cell maintenance solution containing different concentrations of kelp extract was added to the EPC cells, Each group had 8 replicates and cultured at 15°C in a carbon dioxide incubator. At the same time, a normal control group (inoculation of cells, without adding kelp extract, that is, culture with cell maintenance solution) and a blank group (no inoculation of cells, no addition of kelp extract, that is, only adding cell maintenance solu...

Embodiment 3

[0085] Embodiment 3, the protective effect of kelp extract on IHNV infected cells

[0086] 3.1. Measurement method

[0087] 1.0 x 10 EPC cells per well 5 Cells per 100 μL were seeded in 96-well plates and cultured for 24 hours to carry out anti-IHNV effect research. Use cell maintenance solution (2% FBS, 100U / ml penicillin and 100U / ml streptomycin added to MEM medium) to dissolve the kelp extract, and mix with HLJ-15 virus suspension to prepare 0, 100, 200 , 300, 400, 500μg / mL kelp extract mixture. Discard the cell culture medium in the 96-well plate (10% FBS, 100U / ml penicillin and 100U / ml streptomycin were added to the MEM medium), and 100 μL of the above-mentioned mixture was added to the EPC cells, so that the HLJ- The MOI of 15 was 0.01, and each group had 8 repetitions, cultured at 15°C in a carbon dioxide incubator, and observed the cytopathic changes (CPE). At the same time, a positive group (inoculation of cells, inoculation of HLJ-15 (MOI, 0.01) without kelp extr...

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Abstract

The invention belongs to the field of extracts, and particularly relates to preparation and application of an anti-IHNV kelp extract. The invention provides a method for preparing a kelp extract, which comprises the following steps: 1) preparing a kelp crude extract: removing protein in a kelp leaching solution to obtain the kelp crude extract; the kelp leach liquor is an extracting solution obtained by carrying out enzymolysis and water extraction on kelp, and 2) the kelp extract is obtained by carrying out ethanol precipitation on a dialysis retention solution and collecting precipitates, and the dialysis retention solution is in-bag liquid obtained by carrying out dialysis purification on the kelp crude extract by using a dialysis bag method. The invention further provides application of the kelp extract in preparation of an infectious hematopoietic necrosis virus inhibitor or an infectious hematopoietic necrosis virus resisting medicine. The inhibition rate of the kelp extract with the concentration of 300 mu g/mL to IHNV reaches 63.44%.

Description

technical field [0001] The invention belongs to the field of extracts, in particular to the preparation and application of an anti-IHNV kelp extract. Background technique [0002] Salmon is an economical cold-water fish that is mainstreamed at home and abroad today, and it is also one of the high-quality aquatic products with the fastest growing consumer demand. However, the entire industry has been suffering from serious harm from Infectious hematopoietic necrosis (IHN) at present. IHN is an animal disease that must be declared by the World Organization for Animal Health (OIE), and is listed as a second-class animal disease in my country. The disease spreads quickly, is widespread, highly contagious, and difficult to prevent and control. Under natural conditions, virulent strains of Infectious hematopoietic necrosis virus (IHNV) can cause nearly 100% mortality of rainbow trout fry, which has brought huge economic losses to the rainbow trout breeding industry in my country...

Claims

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Application Information

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IPC IPC(8): A61K36/03A61K39/39A61P31/12
CPCA61K36/03A61K39/39A61P31/12A61K2236/19A61K2039/55588
Inventor 任广明徐黎明卢彤岩赵景壮邵轶智
Owner HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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