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Nature feed additive for microorganism-derived aquatic animals, and application of nature feed additive

A technology for feed additives and aquatic animals, applied in animal feed, animal feed, microorganisms, etc., which can solve the problems of fish and shrimp and other aquatic animals with few development and research applications, no safe and effective immune regulatory substances, and lack of acquired immune system and other problems, to achieve the effect of facilitating industrialization, solving expensive costs, and being easy to use and operate

Inactive Publication Date: 2015-06-10
浙江皇冠科技有限公司 +1
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0006] At present, the research on CpG oligonucleotides in the world is mainly in the development of medical applications, and the focus is on the prevention and control of major human diseases and the prevention and control of common vertebrate diseases. There are very few developments and research applications for aquatic animals such as fish and shrimp.
In addition, due to the lack of acquired immune system, invertebrates such as shrimps and crabs mainly rely on biologically active substances produced by living organisms or in the process of biological metabolism or substances extracted from organisms as drugs for the prevention and treatment of breeding diseases. There is no safe and effective drug. Immunomodulatory substance

Method used

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  • Nature feed additive for microorganism-derived aquatic animals, and application of nature feed additive
  • Nature feed additive for microorganism-derived aquatic animals, and application of nature feed additive
  • Nature feed additive for microorganism-derived aquatic animals, and application of nature feed additive

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Experimental program
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Effect test

Embodiment 1

[0039] The preparation of embodiment 1 aquatic animal CpG DNA recombinant plasmid

[0040] (1) Artificially design and synthesize a CpG sequence that has an immune activation effect on aquatic animals, and carry out sulfur modification and polyacrylamide electrophoresis purification (purity 99.99%) to the whole chain of nucleotides. The sequence contains 18 CpG motifs, and an Xho I restriction site is introduced at the 3' end to form a DNA fragment containing 18 CpG motifs. The CpG sequence is shown in SEQ ID NO.1.

[0041] (2) Directionally clone the CpG DNA fragment into the pMD19-T vector, construct the recombinant plasmid pMD19-T-CpG, and transfer it into the DH5α strain for expansion culture; use the alkaline lysis method to extract the plasmid DNA in small batches, and use the agarose gel Electrophoretic detection.

[0042] (3) The recombinant plasmid pMD19-T-CpG was double-digested with homologous enzymes Xho I and Sal I to obtain a CpG DNA fragment with the same cohe...

Embodiment 2

[0046] The transformation of embodiment 2 CpG DNA recombination plasmid and the enlarged culture of engineered bacterium

[0047] (1) The constructed pYES2-5CpG plasmid is electrotransformed into Pichia pastpris GS115 strain, and the electrotransformation parameters are V=1.5kV, 25uF, 200Ohms, 4~10msec; / mL G418), cultured at 30°C until a single colony appeared; it was confirmed that the pYES2-5CpG plasmid had been transferred into Pichia pastoris through plasmid extraction, enzyme digestion and electrophoresis to obtain engineering bacteria;

[0048] (2) Seed culture stage

[0049] The engineering bacteria constructed above were inoculated into 50mL seed medium, cultivated at 30°C and 220r / min for 16h as the primary seed, inserted into 200mL seed medium according to 10% inoculation amount, and cultivated at 30°C and 220r / min for 24h as the secondary seed. grade seeds.

[0050] (3) Initial cultivation stage

[0051] Inoculate the secondary seeds into a fermenter with 3L of ...

Embodiment 3

[0055] Embodiment 3 Aquatic Animal Feed Additives to the Disease Resistance Test of Sea Bass

[0056] Perch juveniles: 7,200 perch juveniles were taken from Zhejiang Ninghai Wanfu Mariculture Co., Ltd. Juvenile fish weigh 20-30g and have a body length of 10-15cm.

[0057] Yeast powder containing CpG DNA recombinant plasmid: provided by Zhejiang Crown Technology Co., Ltd.

[0058] The experiment was divided into 2 groups (test group and control group), each group had 3 repetitions, and 1200 fry were stocked in each repetition. The test group was fed with disease-resistant feed (the amount of yeast powder added was 30 g / kg feed by weight) containing feed additives for aquatic animals (that is, yeast powder containing CpG DNA recombinant plasmids), and the control group was fed with conventional feed (added with normal yeast). Maintain the water temperature at 20-25°C. Feeds were fed normally every day for 4 consecutive weeks, the natural disease mortality was counted, and th...

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Abstract

The invention discloses a nature feed additive for microorganism-derived aquatic animals, and application of the nature feed additive. A preparation method of the nature feed additive comprises the following steps: constructing a recombined engineering bacterium comprising a CpG sequence; carrying out enlarged culture on the recombined engineering bacterium; and collecting thallus, crushing and drying to obtain the nature feed additive for the aquatic animals. According to the preparation method, the CpG sequence is recombined for five times by an isocaudarner technology, and high-copy CpG DNA recombinant plasmid with 90 motifs can be constructed, so that the immune systems of the aquatic animals such as fish and shrimp can be effectively activated, and the nature feed additive has broad-spectrum applicability; a lot of amplification immune stimulation sequences can be obtained by self propagation of the engineering bacterium, so that the problems that the artificial synthesis of the CpG DNA is high in cost and low in production efficiency, is not suitable for large-scale production, and the like can be effectively solved, and the nature feed additive is convenient for industrial popularization; the nature feed additive for the aquatic animals can be directly fed into feed for breeding the aquatic animals, is easy and simple to use and operate, and has the advantages of being good in safety, low in toxicity, high in efficiency, etc.

Description

technical field [0001] The invention relates to the technical field of molecular biology and biological fermentation engineering, in particular to a microbial source natural feed additive for aquatic animals and its application. Background technique [0002] With the rapid development of my country's aquaculture industry, aquatic animal diseases are becoming more and more serious, and various antibiotics, insecticides and other drugs are used in large quantities, which not only threatens the safety of aquatic products, but also causes serious water pollution. Breeding diseases and environmental problems have become one of the important factors restricting the development of aquaculture industry in my country. Taking prawns as an example, viral diseases have broken out in my country since the early 1990s, and there is still no effective control method, which has caused huge losses to the prawn farming industry in my country, and the economic losses caused by the country are a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A23K1/18A23K1/16C12N15/81C12N15/117C12R1/84
Inventor 胡向东叶茂潘玲燕邵建忠
Owner 浙江皇冠科技有限公司
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