| Objective: Antibacterial peptide PR39 is derived from porcine intestinal epithelial cells and belongs to the family antibacterial peptide of cathelicidins.It has a broad-spectrum and high-efficiency antibacterial effect,and has a good inhibitory effect on Gram-positive bacteria,negative bacteria and fungi.At present,the application of PR39 is mainly concentrated in animal husbandry.As a feed additive,it can effectively improve the intestinal function of poultry and enhance the immunity of the body.PR39 can also act as a pro-angiogenic factor to promote the expression of various genes related to angiogenesis.It is conducive to the study of cardiovascular diseases.Screening high-expression PR39 engineering bacteria is a key issue to solve its application in food,medicine and other fields.In this experiment,the principle of synthetic biology was used,and the fluorescent protein was used as a molecular beacon and cis-acting element P2 A as Linker to design a fusion protein expression line,in order to screen non-methanol-induced Pichia pastoris engineering bacteria with high expression of PR39 antibacterial peptide.Methods:(1)This experiment designed two gene lines of fluorescent protein and antibacterial peptide PR-39 fusion expression;(2)using the "biomass" cloning technology in synthetic biology to construct fluorescent protein and antibacterial peptide PR39 Fusion expression cassette;(3)electroporation transformed Pichia pastoris,and then UV-induced mutagenesis,using fluorescent protein as a molecular beacon to screen non-methanol-induced high expression of Pichia pastoris strain.Results:(1)In this experiment,two gene lines were constructed by fusion of green fluorescent protein and red fluorescent protein with multiple copies of PR39 by 2A peptide.(2)The recombinant yeast G-PR39 strain with non-methanol-induced PR39 expression was successfully screened by using fluorescent protein as a molecular beacon,and its expression level was up to 21.8 mg/L;and D-PR39 strain,the expression level was 32.7 mg/L.(3)It was verified by antibacterial experiments that the antibacterial peptide PR39 expressed by recombinant yeast strain had antibacterial activity against Streptococcus faecalis and Escherichia coli.(4)The non-methanol-induced recombinant yeast D-PR39-Y strain was screened by directed mutagenesis,and the expression level was 55.4 mg/L.Conclusion:(1)Fluorescent protein can be used as a molecular beacon to screen high-expression strains intuitively and quickly.Because DsRed2 can be visually observed and GFP requires instrumentation,DsRed2 is more convenient than GFP.(2)The expression level of the recombinant PR39 Pichia pastoris engineering strain G-PR39 constructed in this experiment was 21.8 mg/L,and the expression level of D-PR39 strain was 32.7 mg/L.The expression level of D-PR39-Y strain obtained by mutagenesis screening was 55.4 mg/L,which was 69.4% higher than that of D-PR39.The difference in the expression levels of G-PR39 strain and D-PR39 strain may be due to the difference in genotoxicity of different gene fusion same gene to some extent.(3)The expressed product was proved to have antibacterial activity against Escherichia coli and Streptococcus faecalis by bacteriostatic experiments.(4)The 2A peptide and Biobrik design eliminates the cumbersome steps of cleavage of fusion proteins in vitro,reduces the adverse effects of protein fusion,and increases the screening rate;(5)Using fluorescent protein molecules as molecular beacons and application of 2A peptide,taking into account he advantages of both qualitative and quantitative enable rapid screening of multiple copies by molecular beacons.This experiment lays a solid foundation for further screening for strains with higher expression levels. |
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