Cloning, Prokaryotic Expression And Purification Of Defensin Gene Of Musca Domestica Antibacterial Peptide, And Identification Of Bacteriostatic And Carcinostatic Activities Thereof

Objective The purpose of this study was to perform bioinformatics analysis to thedefensin gene of Musca domestica antimicrobial peptide.This gene was extractedfrom Musca domestica antimicrobial peptide and cloned, expressed and purified;To observe the purified mature defensin (MD) of antimicrobial peptide have theinbibitional effect on bacteria; The inhibitory action of the antimicrobial peptide onthree tumor cells of HepG2,SIHA,SW480was observed;whether or not MD haveinhibitory action on cells of Human Cardiac Myocytes （H9C2);Comparing theanti-proliferative effect of antimicrobial peptide and hydroxyurea on liver cancer cellof HepG2,and judging the anti-tumor effect of antimicrobial peptide is better thanhydroxyurea whether or not.To observe the influence of Musca domestica antimicrobial peptide defensin onHepG2cell cycle;To observe the influence of Musca domestica antimicrobial peptideon cysteinyl aspartic Proteinase-3(caspase-3) of HepG2cell, and we can explore themechanism of MD to Inhibition effect on HepG2cell.MethodsThis part was to predict the immunogenicity of the mature defensin(MD)extracted from Musca domestica antimicrobial peptide encoding by defensingene and characterize its structure by bioinformatics approaches. Thephysico-chemical property, solubility, post-translational modification sites,transmembrane domain,hydrophilicity/hydrophobicity, secondary structure, tertiarystructure,Homology analysis,Genetic relationship comparison,were analyzed andpredicted by using the bioinformatics tools-ExPASy, including ProtParam,SOSUI, TMHMM, HNN, MotifScan, ORF finder, and Bcepred, combined withbioinformatics softwares (Gene Runner, DNAMAN, etc.).The genic sequence of defensin gene was used to design specific primers forcloning mature defensin(MD).RNA was extracted from Musca domestica larva andMD was then amplified by PCR.PCR product was cut with BamHI and XhoI and thenput into pGEX-6P-1vector. Recombinant expression plasmid pGEX-6P-1/MD wasconstructed and target fragment was put into pGEX-6P-1vector stably throughdouble digestion. The resultant recombinant plasmid (pGEX-6P-1/MD)wastransformed into BL21(DE3). The fusion protein was expressed after IPTG inductionat25℃, and purified by GST-Sefinose affinity chromatography.Using methyl thiazolyl tetrazolium (MTT) colorimetric method to find theinhibition rate of tumor cells of SW480,HepG2,SIHA by the effect of differentconcentrations of antibacterial peptide MD, and the inhibition rate to the inhibition ofmyocardial cells.The growth curves of SW480，SIHA，HepG2cells in the above sixdensity groups were made by MTT method and three tumour cells of isometricculture medium were been the control groups.We would find the inhibition rate ofdifferent concentrations of antimicrobial peptides and hydroxyurea acting on HepG2by using MTT colorimetric method，And then find the regression equation and IC50byusing SPSS software according to the concentration and Inhibition rate.If three concentration antibacterial peptides can cause HepG2cells early, mid andlate apoptosis by AnnexinV-Fitc-PI double dye apoptosis detection kit,and isometricculture medium and PBS were been the control groups.Flow cytometry instrumenttest the effect of HepG2cell cycle and apoptosis of three concentration ofantibacterial peptides,and isometric culture medium and PBS were been the controlgroups.Caspase-3enzyme activity was detected by caspase-3detection kit andmicroplate reader. ResultsThrough the bioinformatics found that the protein was composed of92aminoacids,the molecular formula,C435H670N126O124S9; the molecular mass,9.9374ku; thevalue of theoretical isoelectric point,8.75; the absorbance (A) value,0.637(280nm);half-life is30hr.4post-translational modification sites.It is near that Defensin fromMusca domestica and other3kinds of fly defensin of homology.Musca domestica larva antimicrobial peptides MD gene with a molecular size of142bp and the recombinant pGEX-6P-1/MD was successfully constructed. Theresults of SDS-PAGE revealed that the molecular weight of recombinant protein wasapproximately30kDa.The antimicrobial peptides of the Musca domestica larva has inhibitional activityto Escherichia coli.The MTT examination demonstrated that the six densityantimicrobial peptides had all Inhibition activities on SW480，SIHA，HepG2，compared with the normal control group，the difference had significance(P<0.05).The six density groups of antimicrobial peptides had no inhibition activities onHuman Cardiac Myocytes cells， compared With the normal control group,thedifference had no significance(p>0.05).under different concentration of antimicrobialpeptides’ effect, the growth of three kinds of tumor cells of SW480，SIHA，HepG2have obvious inhibition.The higher the drug concentration,The longer theduration,and it will have the higher inhibition to the cells.Regression equation ofantimicrobial Peptides and hydroxyurea were respectively=-1.566+0.529x and=-2.342+0.597x and IC50of that were respectively909μg/ml and8416μg/ml.Between the IC50we could see a big difference which was that IC50valueof antimicrobial Peptides was much smaller than that of hydroxyurea.The effect ofantimicrobial Peptides on inhibition of Proliferation of HepG2cells was greater thaneffect of hydroxyurea on that. The effect of antimicrobial peptides defensin on cell cycle of HepG2cells wasdetected by flow cytometry:The ratio of cells GIPhase and M Phase increased,SPhase decreased.Comparing GI:Phase and S Phase group with the PBS controlgroup,the differences on the ratio of cells had statistical significance(P<0.01),Itillustrated that the antimicrobial Peptide would enable cell cycle block in the GIPhase， thus inhibiting the DNA synthesis of S Phase. The concentration ofantimicrobial peptide group to the Value-added inhibition rate between three tumorcells SW480， SIHA， HepG2have no Significant difference.Caspase-3of theantimicrobial peptide group was significantly higher than that of PBS control groupthere was statistical significance between the two groups.conclusionThrough the bioinformatics we found that Musca domestica larvae antimicrobialpeptides defensin gene has its special structure,maybe it has antimicrobial activityand other biological activity;Plasmid pGEX-6P-1/MD protein was cloned andexpressed successfully. The MD protein was testified which had inhibitory anddamaging effect on HepG2,SW480,SIHA tumor cells. All that aforesaid provided abasis for the researches of antimicrobial peptide drugs from Musca domesticadefensin.The antimicrobial peptides MD of the Musca domestica larva has inhibitionalactivity to Escherichia coli; it has inhibitional effect on SW480,SIHA,HepG2tumorcells by MD.The inhibition proliferation effect of Antimicrobial peptides is betterthan Hydroxyurea to HepG2cells,and it has a great application prospects. With theincrease of drug dose and duration extension that the inhibitional effect on SW480，SIHA，HepG2tumor cells by MD will strengthen.Comparing MD and Chemotherapydrugs,the merit of MD is it can kill the tumor cells,but can’t hurt normal somaticcells;The antimicrobial peptides MD may inhibit tumor cells by two methods:To effect the cell cycle of HepG2by inhibiting the DNA synthesis of S Phase; To induceapoptosis through the channel of mitochondria.