Active Peptide From Acetes Chinensis With Inhibitory Activity On Neuraminidase Of IAV

Neuraminidase is a kind of glycoprotein on surface of influenza virus. It cancut off α-carbohydrate between the residue of NA and host cells. Prompt thefully-developed virus drop from host cells, then transmit virus.So far, most ofthe anti-influenza drugs based on restrain NA activity are via chemical approachsynthetize, such as Zanamivir and Oseltamivir. Obtaining the anti-influenzaagents drugs from natural product is a new way. This text through screen material,determined Acetes Chinensis as the final material. In vitro inhibitory NA activityand MDCK cells infected by IAV test were taken as the indexes.Through isolationtechnique such as SephadexG-15gel chromatography, RP-HPLC（reversed-phasehigh performance liquid chromatography） separate and purify Acetes Chinensiszymolyte,at the same time, combined Spectroscopy（FT-IR）technique to analysethe change of FT-IR in different levels. Obtained highly active polypeptide anddetermined the sequence. Through computer-aided drug design software–SYBYLto imitate the combine of polypeptide and the active region of NA. By chemicalapproach synthetize polypeptide. Through toxicity test, haemagglutination （HA）assay, in vitro inhibitory NA activity to test the properties of polypeptide.Preliminary explored the Structure-activity analysis.The main contents of this paper were as listed:1. Select Acetes Chinesis, Chub, Cod skins, Anchovy as raw materials.digested with protease. Chose Acetes Chinesis as the ideal raw material,throughanalysed amino acid of four materials, speculated amino acid content and typeaffected the inhibitory NA activity. Then tested type of enzyme, enzymolysistime, enzymatic quantity, feed liquid ratio which could affect zymolyte activity.The best condition was: trypsin enzymolysis, enzymolysis time5.5h, enzymatic quantity2000U/g, feed liquid ratio1:7. The inhibitory NA activity IC50at thebest condition was1.32mg/mL. The result showed that it was better thanprevious IC501.86mg/mL.2. Separated and purified Acetes Chinensis enzymatic hydrolysate withSephadexG-15gel chromatography, RP-HPLC（reversed-phase high performanceliquid chromatography）, take inhibit NA activity as index, then obtained a highlya ctive polypeptide. The sequence of polypeptide was EISYIHAEAYRRGELK.hrough computer-aided drug design related software-SYBYL to analog integratesynthetic peptide and NA active region. Preliminary explored the binding sitebetween synthetic peptide and NA active region. Through chemical synthesissynthetize polypeptide. By contrasting the change before and after of theultraviolet spectrum of polypeptide with neuramidinase. We speculated that thesynthetic peptide has occured availably union with neuramidinase.3. By IAV infected the MDCK cells assay, we measured the effect ofpolypeptide to virus, the IC50was0.19mmol/L. The toxicity of the polypeptideto MDCK cells was tested by MTT assay. By observing the morphologicalchange and measured the absorbance values at570nm, obtained the largestnon-toxic concentrations（TC₀） was1.26mg/mL and half of the toxic drugconcentrations(TC₅₀) was7.75mg/mL. Through hemagglutination activity assaywe had a conclusion that virus inhibition rate had the dose dependencyphenomenon when the concentration of polypeptide was125μg/mL-500μg/mL.Then guessed that it existed other target spots in restrain NA activity.