Construction Of Prokaryotic And Eukaryotic Expression System For LfcinB And Other Antimicrobial Peptides

In recent years,the extensive use of antibiotics production and medicine has led to the emergence of super resistant bacteria and brought about many hidden safety hazards.It is urgent to find new drugs that can replace traditional antibiotics.Antimicrobial peptides are cationic polypeptides that are widely present in animals and plants.They have broad spectrums of antibacterial activity and are not easy for pathogens to generate drug resistance.Therefore,antimicrobial peptides have huge potentialas feed additives and food bacteriostatic agents.Bovine lactoferrin is an antibacterial peptide discovered in recent years.It is produced by the hydrolysis of lactoferrin by pepsin and contains only 25 amino acids.The bacteriostatic effect is the strongest among the lactoferrin peptides currently found.With the development of molecular biology and DNA recombination technology,the use of genetic engineering to express antibacterial peptides has become an effective method for the production of antibacterial peptides.Compared with traditional methods,genetic engineering has the advantages of large-scale production,short production cycles,and relatively low costs.In order to make the expression of LfcinB easier to be detected and purified,this experiment fused the red fluorescent protein DsRed2 and His-Tag to the C-terminus of the LfcinB gene,and successfully constructed three plasmids with DsRed2 as the reporter gene: pYES2-?-LfcinB-DsRed2,pYES2-?-LfcinB-DsRed2-HIS and pYES2-?-LfcinB-HIS-DsRed2 are electrotransformed into Saccharomyces cerevisiae W303 and INVSC1.The Oxford cup method was used to detect the inhibitory activity of the induced fermentation broth on E.coli,and the expression products were tested for thermal stability and pH sensitivity;the laser confocal fluorescence microscope was used to detect the fluorescence of yeast cells;and SDS-PAGE electrophoresis was used to detect the protein of interest.The experimental results show that by comparing the changes of OD600 and pH values of the induced and non-induced yeast groups,the induction of expression of the recombinant protein LfcinB-DsRed2-HIS did not adversely affect yeast growth.Using the Oxford Cup method,the fermentation broth was shown to have the activity of inhibiting the growth of E.coli,and proved that induced culture supernatant is resistant to high temperature and sensitive to alkali.Through laser confocal detection,red fluorescence was observed in the induction group,indicating that expression of the recombinant protein was achieved,but SDS-PAGE did not reveal the target protein band in the culture supernatant or the pellet.We presume that the expression level was low.and the causes may be 1.The intracellular expression of antibacterial peptides is still toxic to S.cerevisiae;2.Recombinant protein are not stable,3.Glycosylation may decrease expression level.When purifying the expression product,the position of the target protein was tracked by bacteriostatic activity.As a result,it was found that the ammonium sulfate and acetone precipitation methods could not precipitate the components with bacteriostatic activity in the supernatant of the induced culture solution.Ultra filtration or dialysis causes loss of bacteriostatic activity.We speculate that during the inducible expression of the Saccharomyces cerevisiae strain,the toxicity of the expression product provoked to a stress response in the host cell,which produced a certain small molecule substance with antibacterial activity.Therefore,the antibacterial activity detected by the Oxford Cup method is mainly caused by this small molecule,and the identification of this small molecule needs further research with non-targeted metabolomics strategy.Antibacterial peptides are toxic to prokaryotes and cannot be directly expressed in E.coli.Negatively charged fusion partners are often linked to antibacterial peptides to inhibit their toxicity to E.coli.In organisms,antimicrobial peptides are synthesized in the form of prodomain + antimicrobial peptides.After being digested by a specific protease,the mature antimicrobial peptides then participate in the body's immune response.The prodomain also contains a large number of negative charges.In this experiment,the prodomains or Cherry were used as fusion partners to express in E.coli,and their inhibition effects on antibacterial peptides were investigated.In this experiment,six gene fragments: cherry-piscidin,piscidin-prodomain,sig-piscidin-prodomain,cherry-piscidin-prodomain,piscidin,and prodomain-R-2PLx are ligated to the vector pET22b(+)and transformed and then induced in E.coli BL21(DE3).It was found that the three expression induction of cherry-piscidin,piscidin-prodomain,and cherry-piscidin-prodomain caused E.coli to fail to grow in the early stage of induction,and cherry and prodomain of piscidin could not inhibit their antibacterial activity.The expression products of prodomain-R-2PLx,and sig-piscidin-prodomain did not inhibit the growth of E.coli during the induction period,but no protein bands were detected in the subsequent protein electrophoresis.It is speculated that during translation,mRNA may have a stem-loop structure on the secondary structure.As a result,the protein cannot be translated normally.Relevant experiments will be performed after adjusting the induction conditions.