The Toxicity Research Of The Fusion Polypeptide HEGF-AWRK6

The fusion polypeptide hEGF-AWRK6 is a new antimicrobial peptides.It’s formationed by human epidermal growth factor (hEGF) and antimicrobial peptides AWRK6 fusion,using E. coli expression system according to the codon preferences, designed and expression.The fusion peptide hEGF-AWRK6 has more hydrophilic and strong stability, antimicrobial activity and other characteristics, is a bifunctional peptides,has antibacterial activity and promote healing activity.It has developed into a novel peptide drug potential.Acute toxicity test is a prerequisite for drug development, generally use the mouse oral acute toxicity test, but now the acute toxicity test animal ethics lethal challenge has now become the focus of controversy.Coupled with the animals, "reduce, replace, optimization" put forward 3R principle,Cytotoxicity and establishment of the development model organism toxicity testing model for rapid screening to predict the acute toxicity in rodents and become an international cutting-edge research in the field of toxicity studies.In order to further clinical application of peptide fusion hEGF-AWRK6, this study investigated the toxicity of the fusion polypeptide hEGF-AWRK6 normal cells, model organisms and rodents.In this experiment, first performed vitro toxicity testing and C. elegans toxicity testing,then according to the test results, we estimate the dose acute toxicity test in mice.In order to reduce the use of experimental animals, accurate toxicity test results, exploring vitro alternative toxicology methods.Method:Cytotoxicity test is use normal cells, mouse fibroblasts (3T3) and human epidermal keratinocytes cells (HACAT),test hEGF-AWRK6 fusion peptide cytotoxicity by methods CCK8, concentrations of 5mg/mL, 10mg/mL,20mg/mL, 30mg/mL,50mg/mL,to reflect the effect of hEGF-AWRK6 toxic to cells.On the basis of the cytotoxicity test,use the same concentration gradient fusion peptide hEGF-AWRK6 role for toxicity testing in C.elegans,measure including semi mortality, reproductive ability, athletic ability and longest life.According cell and C.elegans toxicity test results, set the concentration of antimicrobial peptides in mice by intraperitoneal injection toxicity test,mice were injected hEGF-AWRK6 concentrations of 40mg/kg,200mg/kg, 1000mg/kg,2500mg/kg,5000mg/kg.Measure includes half death rates, organ index, sperm deformity rate and blood biochemical indicators.Results:CCK-8 assay cytotoxicity, showed fusion peptide hEGF-AWRK6 has a lower cytotoxic to normal cells,IC50>50mg/mL,when the concentration is greater than 20mg/mL, cell proliferation was inhibited.In C. elegans toxicity test, the fusion peptide hEGF-AWRK6 of MTC=50mg/mL, LC50=22.01mg/mL,95% confidence interval 19.18-25.76,has no significant effect on C.elegans reproductive ability, athletic ability and longest life.Acute toxicity test in mice, MTD> 5000mg/kgBW, LD50> 5000mg/kg BW, based on acute toxicity (LD50) dose grading standards, the fusion polypeptide hEGF-AWRK6 has no actual toxicity,and had no significant effect on organ index in mice, sperm deformity rate, blood biochemical indicators.Conclusion:After experimental testing, we found that fusion polypeptide hEGF-AWRK6 has no actual toxicity.By cytotoxicity, C.elegans toxicity test results were analyzed and found C.elegans toxicity study model is an alternative to traditional rodent toxicity studies of the feasibility of the method.