Expression Of A Recombinant Hybrid Antimicrobial Peptide In The Mycelium Of Cordyceps Militaris And Its Function Validation

Antibiotics,with the efficacy of growth promotion and disease prevention,are used widely in livestock and poultry production.However,the abuse of them in recent years has led to the hidden dangers of antibiotic residues and the emergence of drug-resistant bacteria.With the increase of consumers'demand for“non-resistant”products,the development of safe and efficient alternatives to new feed antibiotics has become a hot issue in the present research of healthy breeding of livestock and poultry industry in China.As an alternative to antibiotics,antimicrobial peptides have the characteristics of broad-spectrum antibacterial activity,immunomodulatory activity and little drug resistance.Among them,Magainin II isolated from Xenopus laevis skin and Cecropin B isolated from Bombyx mori are considered good candidate molecules for enhancing resistance to bacterial diseases.In addition to the naturally-occurring AMPs,many recombinant AMPs have been designed to achieve increased stability and specificity.However,Cordyceps militaris,the edible and medicinal fungus,has not been reported as a vehicle for AMP production.Based on these potential advantages,in the present study,we aimed to express the hybrid antimicrobial peptide gene Magainin II-Cecropin B(Mag II-CB)and the single cecropin B(CB)gene,in C.militaris mycelium.His-tagged Mag II-CB and CB recombinant proteins were purified using Ni-NTA Sefinose resin His-tagged Columns.The antibacterial function and mechanism of the AMPs was evaluated in vitro using antibacterial assays.We also investigated the immunomodulatory activities of the purified recombinant hybrid AMPs and freeze-dried C.militaris producing AMPs after their administration to BALB/c mice infected with Escherichia coli(E.coli,ATCC 25922).Our findings suggest that both purified recombinant AMPs and C.militaris mycelium producing AMPs display antibacterial and immunomodulatory activities.The obtained results in this study are as follows:1.The optimal conditions for protoplast extraction of C.militaris are as follows:the mycelium age of 4 d,the enzymolysis temperature of 34?and the time of 4 h.The extraction rate of protoplasts of C.militaris reached 10~7-10~8 cells/mL.The regeneration medium for the protoplasts of C.militaris was PDA medium containing 0.8 M mannitol and the minimum inhibitory concentration of hygromycin B resistance was 650 mg/L.The optimal conditions for Agrobacterium tumefaciens transformation are as follows:the acetosyringone concentration of200?M,the co-culture time of 2 d and the number ratio of A.tumefaciens to C.militaris protoplasts of 10~7/10~5.2.The binary T-DNA expression vector,which contains the hybrid antimicrobial peptide gene Mag II-CB and the single CB gene,was successfully constructed.Agrobacterium tumefaciens was then separately transformed with the pCB130-Mag II-CB and pCB130-CB plasmids.PCR and Western Blot showed that 6 strains of the Mag II-CB transformed C.militaris and 5 strains of the CB transformed C.militaris.The ELISA assays showed that the highest contents for Mag II-CB and CB were 0.938±0.072‰and 0.705±0.054‰of total soluble proteins,respectively.3.His-tagged Mag II-CB and CB recombinant proteins were purified using Ni-NTA Sefinose resin His-tagged Columns.The purities of the Mag II-CB and CB proteins were 82.6%and 80.4%,respectively,as judged by ELISAs.The recombinant Mag II-CB exhibited more effective antibacterial activities than CB in terms of its ability to inhibit Gram-negative and Gram-positive bacteria by measuring the diameter of the agar plate bacterial diffusion clearance zone and the minimal inhibitory concentrations(MICs)of them.In addition,the effects of recombinant AMPs on the cell membrane permeability of E.coli and Staphylococcus aureus by scanning electron microscopy were analyzed.The results showed that the Mag II-CB and CB could act on the bacterial cell membrane by extracellular antibacterial mechanism,eventually leading to cell death,and the recombinant antimicrobial peptide Mag II-CB showed a stronger antibacterial effect than CB.4.Based on the purified recombinant antimicrobial peptides,the mice infected with E.coli ATCC 25922 were used to determine the minimal lethal dose for this bacterium,and the minimal doses of Mag II-CB and CB required to protect the mice from infection were 1×10~9CFU/mL and 8 mg/kg,respectively.Mag II-CB and CB could regulate the changes in intestinal microbial flora,improve intestinal villus in mice,and alleviate intestinal villus length,crypt depth and V/C value.Mag II-CB maintained the integrity of the intestinal mucosal barrier by up-regulating tight junction proteins(zonula occludens-1,claudin-1 and occludin).Mag II-CB treatment also supported immune functioning in the mice by regulating their plasma immunoglobulin(IgA,IgM,IgG)and ileum secreted immunoglobulin A levels,by attenuating their pro-inflammatory cytokine(IL-6,MCP-1 and TNF-?)levels,and by elevating their anti-inflammatory cytokines IL-10 level.5.The mice infected with E.coli(ATCC 25922)were directly fed with the C.militaris mycelium producing AMPs to further investigate its antibacterial and immunomodulatory activities.The results showed that directly feeding the mice with the C.militaris mycelium producing Mag II-CB and CB could maintain the integrity of the intestinal morphology by modulating the intestinal microbial flora and improving intestinal mucosa and villus damage.In addition,Mag II-CB could up-regulate tight junction proteins(zonula occludens-1,claudin-1and occludin)and down-regulate plasma immunoglobulin(IgA,IgM,IgG)and ileum secreted immunoglobulin A levels.Moreover,Mag II-CB decreased the pro-inflammatory cytokine(IL-6and TNF-?)levels,and increased anti-inflammatory cytokines IL-10 level,which was consistent with the results of the research on purified recombinant AMPs.