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A kind of yeast engineering bacteria producing specific antimicrobial peptide and its fermentation application
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A technology of yeast and Saccharomyces cerevisiae, applied in the fields of biotechnology and genetic engineering, can solve the problems of complex extraction process, difficult to obtain, and high cost of chemical synthesis
Active Publication Date: 2019-10-29
GUANGDONG RONGDA BIOENG CO LTD +1
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Problems solved by technology
Due to the low expression of natural antimicrobial peptides and the complicated extraction process, it is difficult to obtain large quantities; and the cost of chemical synthesis is high, expensive, and has certain toxicity to most microbial hosts, which also leads to large-scale production and application of antimicrobial peptides has been constrained
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Embodiment 1
[0097] Example 1, the acquisition of specific antimicrobial peptides and their coding genes
[0098] After analyzing and optimizing a variety of antimicrobial peptide sequences in various species, an antimicrobial peptide gene was obtained, which was named mor gene. The base sequence of mor gene is shown in sequence 1 of the sequence table. The peptide is named mor antibacterial peptide, and its amino acid sequence is shown in sequence 2 of the sequence list.
Embodiment 2
[0099] Embodiment 2, construction of multi-copy recombinant vector pUGrx-mor
[0100] 1. Construction of recombinant vector pGAPZαA-mor
[0101] 1. Artificially synthesize the DNA molecule shown in sequence 1 of the sequence listing.
[0102] 2. Using the DNA molecule obtained in step 1 as a template, PCR amplification is performed using primer F1 and primer R1 to obtain a PCR amplification product.
[0105] In F1, the underlined part is the EcoR I restriction site and protective base;
[0106] In R1, the underlined part is the Xba I restriction site and the protective base.
[0107] 3. The PCR amplified product of step 2 was double-digested with restriction enzymes EcoR I and Xba I, and the digested product was recovered.
[0108] 4. Digest the pGAPZαA plasmid with restriction endonucleases EcoR I and Xba I to recover a vector backbone of about 3100 bp.
...
Embodiment 3
[0132] Embodiment 3, the acquisition of Saccharomyces cerevisiae engineering bacteria pm148
[0133] 1. Construction of Saccharomyces cerevisiae engineered strain pm148
[0134] 1. Transform Saccharomyces cerevisiae with the recombinant vector pUGrx-mor obtained in Example 2 to obtain recombinant bacteria.
[0135] 2. The recombinant bacteria obtained in step 1 were subjected to G418 resistance screening to obtain several strains of multi-copy integrated positive recombinant bacteria that could grow on YPD medium plates containing G418 at a concentration of 1000 μg / mL.
[0136] 3. The pSH65 plasmid is transformed into each bacterial strain obtained in step 2 to obtain recombinant bacteria.
[0137] 4. Inducing and culturing the recombinant bacteria obtained in step 3 in the YPG liquid medium containing galactose to express Cre enzyme, recognize the loxP site, and obtain the recombinant bacteria excising the resistance gene KanMX.
[0138] 5. After completing step 4, each rec...
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Abstract
The invention discloses a yeast engineering bacterium producing antimicrobial peptides and its fermentation application. The present invention provides Saccharomyces cerevisiae pm148, whose preservation number is CCTCC NO: M2016761. The present invention uses safe nutritional Saccharomyces cerevisiae as a host, adopts the constitutive promoter GAP as a regulatory factor, and uses rDNA as an integration site to construct a Saccharomyces cerevisiae engineering strain without exogenous resistance gene fragments and high biological safety. Multiple copies of the promoter are integrated to efficiently start the expression of antimicrobial peptides without induction or purification. The fermentation product adopts a low-temperature spray-drying process to directly prepare yeast preparation products rich in antimicrobial peptides. This product has good antibacterial properties, especially for the large intestine Bacillus and other Gram-negative bacteria have a significant antibacterial effect, and can be used directly as a feed additive with good application effect.
Description
technical field [0001] The invention relates to the fields of biotechnology and genetic engineering, in particular to a yeast engineering bacterium producing antimicrobial peptides and its fermentation application. Background technique [0002] The emergence of antibiotics plays an important role in human defense and treatment of bacterial infectious diseases, and has made great contributions to human health. However, with the wide-scale application of antibiotics, especially at the level of agriculture and aquaculture, the overuse of antibiotics has brought many adverse effects on food safety, ecological safety and human health. It is necessary to find suitable alternatives to antibiotics and develop them without harmful residues in the body. , Can not or is not easy to produce drug resistance, the research of effective alternatives to antibiotics that are green and environmentally friendly is becoming a current hot topic and has received widespread attention. [0003] Ant...
Claims
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