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A kind of protein for improving the ability of sturgeon to resist pathogenic bacteria and its preparation method and application

A protein and sturgeon technology, applied in the field of protein engineering, can solve problems affecting aquatic product quality and human health, drug residual water environment, pathogen resistance, etc., achieve good immune regulation, improve efficiency and economic benefits, and improve effect of immunity

Active Publication Date: 2022-07-08
BEIJING FISHERIES RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, drug prevention and control is the main measure for the treatment of aquatic diseases. In the disease prevention and control technology of sturgeon, the common method is to directly add finished antibiotics to the processed feed. Although the drugs can prevent and control diseases to a certain extent in a short period of time, but It will also bring some disadvantages, such as long-term frequent use of antibiotics and some other chemical drugs will cause drug resistance of pathogens, drug residues in fish and pollution of water environment, etc., also affect the quality of aquatic products and human health

Method used

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  • A kind of protein for improving the ability of sturgeon to resist pathogenic bacteria and its preparation method and application
  • A kind of protein for improving the ability of sturgeon to resist pathogenic bacteria and its preparation method and application
  • A kind of protein for improving the ability of sturgeon to resist pathogenic bacteria and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Determination and cloning of differentially expressed genes with changes in immune function under different conditions

[0038] In order to study whether conditions such as immunogen stimulation or high temperature stress affect the immune function of sturgeon, the inventor's team has sequenced the liver transcriptome of sturgeon under different conditions. expressed genes.

[0039] The primers were designed according to the sequence, and the Siberian sturgeon spleen cDNA was used as the template to amplify the partial fragment of the cDNA. Specific primers GSPI, NP3, GSPII and NP5 were designed according to the obtained cDNA fragment sequence for RACE assay.

[0040] Abirp-GSP5-1:5'-CCGCTGCAGGACAGGAAGTAATTAC-3'; SEQ ID NO. 7

[0041] Abirp-GSP5-2: 5'-TGTAGGTGGAGATGAGGGGAGTCACT-3'; SEQ ID NO. 8

[0042] Abirp-GSP3-1:5'-TAACGGAAGAAAGGACCCAAGACGCG-3'; SEQ ID NO. 9

[0043] Abirp-GSP3-2: 5'-ATAAGGAGATCTGTGATGAACTCAGA-3'; SEQ ID NO. 10

[0044] The total RNA ...

Embodiment 2

[0045] Example 2 Preparation of Siberian sturgeon IRP recombinant protein

[0046] 1. Cloning of the Siberian sturgeon IRP mature peptide: According to the gene sequence obtained in Example 1, the SignalPv5.0 Server software was used to analyze the signal-free peptide online, and the Siberian sturgeon spleen cDNA was used as a template to design with NcoI and HindIII restriction sites respectively. The primers F(F:C CCATGGCTCTGCAGCCTCTCGACATC, SEQ ID NO. 5) and R (R: CC AAGCTT CTAAACAGGGGGGGC, SEQ ID NO. 6) was subjected to PCR amplification to amplify the cDNA fragment of the Siberian sturgeon IRP mature peptide of amino acids 3-143.

[0047] The 25 μL reaction system contains 2.5 μL 10×Ex buffer, 2 μL dNTP, 0.3 μmol each of F and R primers, 1 U Extaq enzyme (Takara), and 500 ng cDNA template. The PCR reaction program is: 94 °C for 10 min; 72°C for 30s (10 cycles); 94°C for 25s, 60°C for 25s, 72°C for 30s (25 cycles); 72°C for 7min. After analysis by 1% agarose gel elec...

Embodiment 3

[0068] Example 3 Expression characteristics of Abirp in different tissues of Siberian sturgeon

[0069] Real-time quantitative PCR was used to detect the relative expression of Abirp in different tissues of healthy Siberian sturgeon, and to study the tissue distribution of AbirpmRNA.

[0070] Specific operation method: randomly select 11 tissues of 10 healthy Siberian sturgeon (weight about 150g) of 10 1-year-old males and half, respectively: blood, gills, eyes, head kidney, spleen, heart, muscle, skin, liver, brain and intestines. Total RNA was extracted by Trizol method and reverse transcribed into cDNA by Takara reverse transcription kit.

[0071] According to the cDNA sequence of Abirp, the primers for fluorescence quantitative PCR were designed. The primer sequences are as follows:

[0072] AbirpF: 5'-GCCCAACTCACCCGTCCT-3';

[0073] AbirpR: 5'-GGTTTATGTTGCTGTCTCGCTTGT-3';

[0074] The primer sequences of the internal reference gene 18S rRNA are as follows:

[0075] 1...

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Abstract

The invention relates to a protein for improving the ability of sturgeon to resist pathogenic bacteria, a preparation method and application thereof. The amino acid sequence of the protein that can improve the ability of sturgeon to resist pathogenic bacteria provided by the present invention is shown in SEQ ID NO.2. By sequencing the liver transcriptome of sturgeon under different conditions, a gene differentially expressed under different conditions with changes in immune function was found according to the sequencing results, and the total RNA of the spleen of sturgeon was extracted, and the full-length cDNA sequence was obtained by splicing using RACE technology. As shown in SEQ ID NO. 1, the recombinant protein AbIRP was obtained by recombinant expression technology. It was found that the injection of recombinant protein AbIRP had significant clearing and inhibitory effects on sturgeon-infected pathogens. As an immune adjuvant, it could improve the immune effect of the vaccine. Feed additives can improve the disease resistance of hybrid sturgeon to pathogenic bacteria.

Description

technical field [0001] The invention relates to the technical field of protein engineering, in particular to a protein for improving the ability of sturgeon to resist pathogenic bacteria and a preparation method and application thereof. Background technique [0002] Sturgeon is a cartilaginous scaly species, which is easy to domesticate, grows fast, and has high economic value. It is an important aquaculture species in my country. In recent years, with the expansion of the scale of sturgeon farming, high-density farming often leads to frequent occurrence of diseases, which brings huge losses to the aquaculture industry. During the whole sturgeon breeding period, the most harmful and deadly diseases were mainly sturgeon septicemia caused by Aeromonas hydrophila and mixed enteritis caused by Aeromonas mildew. [0003] At present, drug prevention and control is the main measure for the treatment of aquatic diseases. In the disease prevention and control technology of sturgeon,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/46C12N15/12C12N15/70C12N15/66A61K38/17A61K39/39A61P37/04A61P31/04A23K50/80A23K20/147A23K20/195A23K20/153
CPCC07K14/461C12N15/70C12N15/66A61K39/39A61P37/04A61P31/04A23K50/80A23K20/147A23K20/195A23K20/153A61K38/00A61K2039/55516Y02A40/81
Inventor 王晓雯朱华胡红霞张蓉刘丽丽
Owner BEIJING FISHERIES RES INST
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