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
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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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