Tandem Expression And Biological Activity Determination Of Antimicrobial Peptide MAF-1A From Musca Domestica

Objective: To construct a prokaryotic expression system of 5 multimeric Musca domestica antifungal peptide-1A(5×MAF-1A)gene,tandem express MAF-1A in Escherichia coli and determine the antimicrobial activity of MAF-1A monomer against Candida albicans in vitro.To investigate the activity and mechanism of MAF-1A against Human Hepatocellular carcinoma cells(HepG2).Methods: 1.The tandem gene encoding MAF-1A was optimized and synthesized with reference to the codon preference of E.coli.After that,the 5×MAF-1A gene was cloned into prokaryotic expression vector pET-28 a.The recombinant vector was transformed into E.coli Roseeta(DE3)competent cells and the expression of recombinant antimicrobial peptide was induced by IPTG.The expression products were analyzed with SDS-PAGE electrophoresis and Western Blot.2.The recombinant 5×MAF-1A protein was purified by Ni-NTA.After 5×MAF-1A protein cleaved specially with enterokinase(EK),MAF-1A monomer was obtained.3.The antimicrobial activity of MAF-1A monomer against C.albicans in vitro was determined by K-B agar diffusion method and microdilution method.4.HepG2 cells and Hepatic stellate cells(HSC)were treated with different concentrations of MAF-1A monomer(12.5μg/mL、25μg/mL 、 50μg/mL 、 100μg/mL 、 200μg/mL),respectively.The proliferation inhibition of HepG2 and HCC cells were detected by inverted microscope morphological observation at different time after treatment.Meanwhile,HepG2 cells and HSC cells,which were not treated with MAF-1A,were used as growth controls.5.The morphological changes of the cells were observed by inverted microscope.The proliferation inhibition and the apoptosis in vitro were detected by MTT assay,LDH kit,flow cytometry and transmission electron microscopy.Results: 1.The expressive vector has been constructed and confirmed by restriction enzyme digestion and DNA sequencing analysis.SDS-PAGE analyses spotted the recombinant 5×MAF-1A protein was successfully expressed in E.coli.The target protein was highly expressed in E.coli under the conditions of 0.8 mmol/L IPTG,28 and 12 hours.℃ 2.The results of recombinant protein purified and cleaved specially showed that we obtained the 5×MAF-1A protein and MAF-1A monomer.The concentration of MAF-1A monomer was 2.0mg/mL.3.The results of Antifungal test in vitro showed that MAF-1A monomer had anti-Candida albicans activity.The MIC and MBC of C.albicans were 0.5 mg/mL and 1.0 mg/mL.4.MTT assay results showed that the inhibitory and cytotoxicity effect on HepG2 cells became more and more obvious with the increasing concentration of MAF-1A.In addition to high concentrations of MAF-1A,the inhibitory effect and cytotoxicity to HSC cells was not obvious.5.LDH test analyses found that MAF-1A can cause massive release of LDH in cells.The results of flow cytometry and transmission electron microscopy suggested that the MAF-1A can inhibit proliferation and induce apoptosis of HepG2 cells,which is mainly observed in early stage of apoptosis.Transmission electron microscopy analysis revealed the nucleus of HepG2 cells was round,chromatin aggregation or edge set,nuclear pyknosis,cytoplasm concentration.Conclusion: 1.In this experiment,we have successfully constructed the 5×MAF-1A prokaryotic tandem expression system,The fusion antimicrobial peptide 5×MAF-1A was further highly expressed in E.coli.2.After the fusion antimicrobial peptide 5×MAF-1A was purified and cleaved,we obtained a high purity MAF-1A monomer,which has the activity to against C.albicans.3.We suggested MAF-1A can inhibit the growth and proliferation of HepG2 cells by destroying the cell membrane and inducing apoptosis.