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
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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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