New method for fast selecting engineering bacteria of leaven of efficient expression antimicrobial peptides

Abstract

The invention provides a new method for fast selecting engineering bacteria of leaven of efficient expression antimicrobial peptides. The method comprises the following steps: 1. constructing plasmidsfor syncretizing antimicrobial peptide genes and chaperone protein genes; 2. carrying out enzyme restriction for linearizing the plasmids; 3. transforming the plasmids to pichia competent thalli through electroporation, utilizing auxotroph for selecting, and cultivating the plasmids for 36 to 48 hours at the temperature of 28 DEG C; 4. directly obtaining transformants from a prescreening plate, i.e. obtaining the engineering bacteria of the leaven of the efficient expression antimicrobial peptides primarily, and then the engineering bacteria of the leaven can be obtained from large number oftransformants through reselection. The invention has simple experiment process and easy operation, the entire screening process can be shortened by 5 to 6 days, and the expenses of labor, materials and finical resources are greatly reduced. The invention is suitable for the engineering bacteria expressed in the leaven for proteins of the antimicrobial peptides, enzyme, antibodies, polypeptide andthe like which is not easy to or can not adopt a functional approach for selection.

Description

technical field [0001] The present invention relates to high-efficiency expression and screening technology of bioengineering bacteria, and is especially suitable for expression products such as polypeptides and proteins that cannot be directly screened by function, or for expression products that are very complicated and require a lot of time to screen. It is especially suitable for large-scale, short-term, large-scale screening of yeast engineering bacteria that highly express antimicrobial peptides. Background technique [0002] Antimicrobial peptides are a kind of peptide substances produced by the host defense system to resist external pathogen infection, and are an important part of the host immune defense system. Antimicrobial peptides not only have a broad antibacterial spectrum, but also have the effect of inhibiting the growth of cancer cells and are not easy to produce drug resistance, which makes the development and utilization of antimicrobial peptides a recent ...

AI Technical Summary

Problems solved by technology

Yeast secretion vectors generally have genes encoding these antibiotics. Since yeast secretion vectors are integrated into chromosomes, in theory, if yeast can decompose high concentrations of antibiotics, then there will be multiple copies of secretion expression vectors on the chromosomes. , which may be high-yielding bacteria, but studies have shown that yeast that can grow on high-concentration antibiotics does not directly correspond to high-expressing yeast, that is to say, although the copy number is high, it can decompose high-concentration antibiotics. It must be a high-yielding bacterium of the target product; our research has also confirmed this conclusion. In the experiment, we screened a strain of yeast engineered bacteria whose resistance was increased by 10 times, but the potency of antimicrobial peptides did not increase

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