A kind of yeast engineering bacteria of fish natural killer cell enhancing factor and its application

A natural killer cell and yeast engineering technology, which is applied in the field of fish natural killer cell enhancer yeast engineering bacteria, can solve the problems of limited drug use effect, no drug available, and drug resistance of pathogenic bacteria, so as to improve the survival rate and Effects on autoimmunity, protection of food safety and human health, and major social and economic benefits

Active Publication Date: 2021-01-05
杭州皇冠农业生物工程技术研究中心有限公司
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  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Long-term drug abuse not only limits the effect of drug use, delays treatment, and causes a large number of deaths and double losses of drug input; The lessons of excessive residues in farmed animals are extremely painful, and the farming industry relies on the depletion of certain ecological resources for sustainable development.

Method used

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  • A kind of yeast engineering bacteria of fish natural killer cell enhancing factor and its application
  • A kind of yeast engineering bacteria of fish natural killer cell enhancing factor and its application
  • A kind of yeast engineering bacteria of fish natural killer cell enhancing factor and its application

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Embodiment 1

[0032] (1) Acquisition of NKEF gene from puffer fish

[0033] About 100 mg of the spleen tissue of the blue-spotted puffer fish was taken, and the tissue was ground into powder in liquid nitrogen, and the total RNA was extracted with the Trizol kit of Invitrogen Company, and the extraction method was carried out according to the instructions. The total RNA was then reverse-transcribed into a cDNA template using the cDNA Synthesis Kit (M-MLV Version), a reverse transcription kit from TaKaRa;

[0034]According to the NKEF gene sequence of blue-spotted puffer fish (Genbank No. DQ003333.1), the expression vector was designed to construct primers P1 and P2, and the EcoRI restriction site was introduced upstream from before the start codon ATG, and downstream from the stop codon TAA Introduce XhoI restriction site;

[0035] P1: 5'-GGGAATTCAAAATGGCTCAGGC-3' (-sense with EcoRI)

[0036] P2: 5'-CCGCTCGAGTTAGTGCTTGGAGAAG-3' (-sense contains XhoI)

[0037] Take the reverse transcripti...

Embodiment 2

[0065] The construction method of yeast engineering bacterium comprises the following steps:

[0066] 1) According to the NKEF gene sequence Genbank No.DQ003333.1 of the blue-spotted puffer fish registered in the GeneBank database, an expression vector was designed to construct primers P1 and P2:

[0067] P1: 5'-GGGAATTCAAAATGGCTGCAGGC-3' (-sense with EcoRI)

[0068] P2: 5'-CCGCTCGAGTTAGTGCTTGGAGAAG-3' (-sense with XhoI)

[0069] 2) Use the Trizol kit to extract total RNA from the spleen of puffer fish, and use the reverse transcription kit cDNA SynthesisKit (M-MLV Version) to reverse transcribe the total RNA into a cDNA template;

[0070] 3) Using the puffer fish cDNA sequence obtained in step 2) as a template, the NKEF gene fragment was obtained by PCR amplification;

[0071] PCR system: 10×PCR Buffer 2μl, dNTP Mixture 0.4μl, Primer1 and 2 each 0.8μl, cDNA template 0.2μl, Ex Taq polymerase 0.2μl, add ddH2O to make up to 20μl;

[0072]PCR parameters: pre-denaturation at 94...

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Abstract

The invention discloses engineered yeast of a fish natural killer cell enhancer and an application thereof. The puffer fish NKEF engineered yeast is named as saccharomyces cerevisiae INVSC1-pYES2-NKEFwith the preservation number of CGMCC1847. The engineered yeast is obtained by inserting the puffer fish NKEF gene into the place between EcoRI and XhoI restriction enzyme cutting sites of a pYES2 carrier to construct a recombinant plasmid and then introducing the recombinant plasmid into the expression host saccharomyces cerevisiae. The engineered yeast is cultivated under the condition of 30 oC, induced by 2% galactose, and the expression of NKEF recombinant protein can reach more than 20%. The anti-disease application of the above-mentioned engineered yeast in aquatic animal breeding is also disclosed, and the effect of improving the anti-infective capability and autoimmune capability of aquaculture animals can be achieved.

Description

technical field [0001] The invention relates to the fields of aquatic biology technology and aquaculture disease prevention and control, in particular to a yeast engineering bacterium of fish natural killer cell enhancing factor and its application. Background technique [0002] With the rapid development of aquaculture industry, disease has become one of the major restrictive factors for the development of aquaculture industry. [0003] Long-term drug abuse not only limits the effect of drug use, delays treatment, and causes a large number of deaths and double losses of drug input; The lessons of excessive residues in farmed animals are extremely severe, and the farming industry relies on the depletion of certain ecological resources for sustainable development. Especially after my country's accession to the WTO, the requirements and standards for the environment and food hygiene and safety have become higher and higher, a large number of antibiotics have been banned, and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81A23K10/16A23K50/80C12R1/865
CPCA23K10/16A23K50/80C07K14/461C12N15/81
Inventor 胡向东叶茂胡伟卿
Owner 杭州皇冠农业生物工程技术研究中心有限公司
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