Preparation method and application of recombinant Ctenopharyngodon idella interferon-interleukin 1 bigeminal protein

A technology of catenin and interferon, which is applied in gene recombination methods and application fields, can solve the problems of fish damage to the water environment, restrictions on popularization and application, instability, etc., and achieve good economic benefits and market prospects

Inactive Publication Date: 2014-07-23
NANCHANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The chemical method is basically banned because it may cause damage to the fish body and cause large-scale pollution or even damage to the water environment; the antibiotic method only has a good effect on bacteria but cannot kill viruses, and it will cause fish residues. use it with caution
Although the vaccine method is effective, it is unstable, which greatly limits its popularization and application

Method used

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  • Preparation method and application of recombinant Ctenopharyngodon idella interferon-interleukin 1 bigeminal protein
  • Preparation method and application of recombinant Ctenopharyngodon idella interferon-interleukin 1 bigeminal protein
  • Preparation method and application of recombinant Ctenopharyngodon idella interferon-interleukin 1 bigeminal protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The preparation method of the recombinant grass carp interferon-interleukin 1 dicatenin.

[0022] (1) Grass carp IL-1 Gene cloning.

[0023] A pair of primers were designed according to the known cDNA sequence of grass carp IL-1 (GenBank accession No. JX014320) Ci IL-F(tat ccatgg ctatggcatgcgaacg) (including Nco I) and Ci IL-R(tca ggatcc cttgttctccagtgtg) (including Bam H I), use purified cDNA as template for PCR amplification reaction, reaction system: 10 × Ex Taq Buffer 2.5 μL, dNTP (2.5 mM) 1.0 μL, primers 0.5 μL, cDNA 1.0 μL, TaKaRa Ex Taq polymerase (5 U / μL) 0.25 μL, add sterilized ddH2O to 25 μL. The reaction conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 1 min, and extension at 72°C for 10 min. After the reaction, the PCR product purification kit was used to purify the amplified product, and the purification effect was detected by 1.0% agarose gel...

Embodiment 2

[0035] Potency determination of recombinant grass carp IFN-IL-1 protein.

[0036] (1) Titer determination of grass carp hemorrhagic disease virus (GCHV): The titer of GCHV was determined by the conventional half tissue culture infectious dose assay (50% tissue culture infectious dose assay, TCID50).

[0037] (2) Determination of potency: Inoculate CIK cells in a 96-well cell culture plate, culture at 28°C for 15 h, grow and multiply until a single layer of cells covers the bottom of the well, and use the initial protein concentration of 750 μg / mL in Example 1 The purified recombinant protein was serially diluted 10 times to stimulate the cells, and each dilution gradient was set up with 8 replicate wells, and cultured at 28°C for 6 h, and then 1.0 × 10 4.5 TCID 50 CIK cells were infected with GCHV / mL dose, and the cytopathic effect (CPE) appeared in the virus-infected control group cells (without protein treatment). In the dilution series, the reciprocal of the highest dilu...

Embodiment 3

[0039] Immunoprotective effect of recombinant grass carp IFN-IL-1 against grass carp hemorrhagic disease virus GCHV.

[0040] (1) Protection of recombinant grass carp IFN-IL-1 on CIK cells.

[0041] A. Preparation of MTT working solution: Weigh 50 mg of MTT powder, fully dissolve it in 10 mL of PBS (0.01 mol / L, pH7.4), operate aseptically with a 0.22 μm microporous filter, and filter to sterilize.

[0042] B. Collect CIK cells, adjust the concentration of the cell suspension, add 100 μL to each well, and adjust the density of the cells to be tested to 1000-10000 / well (the edge wells are filled with sterile PBS).

[0043] C. Cultivate in a 28°C incubator until the monolayer of cells on the 96-well flat bottom plate covers the bottom of the well, add the purified protein solution in Example 1 with a series of 10-fold dilution concentration gradient (10-1~10-8): 100 per well μL, set up 8 replicate wells. At the same time, a negative control group was set up. Continue to cultur...

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Abstract

The invention relates to a preparation method and an application of a recombinant Ctenopharyngodon idella interferon-interleukin 1 bigeminal protein. The method is characterized in that the method comprises the following steps: 1, cloning grass carp IL-1 gene; 2, constructing a combined expression vector Pet-32a(+) / CiIFN-IL-1 containing IFN and IL-1; and 3, expression and purification of the recombinant bigeminal protein: converting the combined expression vector Pet-32a(+) / CiIFN-IL-1 to Escherichia coli BL21(DE3)pLysS, carrying out IPTG induced expression of the recombinant protein IFN-IL-1, and purifying the obtained fusion protein through Ni-NTA affinity chromatography. The bigeminal protein can simultaneously have the immune efficacies of IFN and IL-1, overcomes the disadvantages of respective addition of IFN and IL-1 and accurate proportions thereof in a feed, and is an efficient non-toxic Ctenopharyngodon idella disease resistance preparation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to gene recombination methods and applications. technical background [0002] Grass carp has tender meat and delicious taste. It has the characteristics of simple food habit, wide source of bait, rapid growth and obvious economic benefits. In recent years, the scale and level of grass carp farming in our province have been greatly improved, and its farming output has leapt to the first place, making a great contribution to the prosperity of the local rural economy. However, with the development of high-density farming, grass carp suffers from various diseases during the entire growth period from fry to adult fish, especially viral diseases and bacterial diseases, which have become a bottleneck restricting the healthy development of grass carp. There are many ways to prevent and control fish diseases, including chemical methods, antibiotic methods and vaccine methods. The chemical method...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70A61K38/21A61K38/20A61P31/12
Inventor 胡成钰吴初新李东明
Owner NANCHANG UNIV
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