Separation And Purification Of Antibacterial Peptide From Hermetia Illucens And The Effects Of Active Ingredient On The CNE2 Cells

Objective: To separated and purified antimicrobial peptide from the larvae hemolymph of Hermetia illucens,and singled out the active ingredient.To investigate the effects and mechanisms of the antimicrobial peptide active ingredient on nasopharyngeal carcinoma(CNE2)cells for providing the theory basis that excavating the medical value of research and exploitation Hermetia illucens antibacterial peptide.Methods:(1)The crude extraction of antimicrobial peptides was obtained by the method of induced with Staphylococcus aureus acupuncture;The Tricine-SDS-PAGE electrophore sis was used to detect the existence of small molec ule antibacterial peptides in hemolymph after the induction of larvae of Hermetia illucens;Antibacterial effect of crude extraction of antimicrobial peptide was checked by drug sensitive slips method;The RP-HPLC method was used to isolate and purify the crude extractions of the antimicrobial peptide,and the corresponding components of the purified peaks were collected;The antibacterial effect of the corresponding components of each purified peak was measured to screen out a single component with antibacterial activity,and the MIC value of the active component was measured.(2)The CCK-8 method was used to detect the effect of each component on the proliferation of nasopharyngeal carcinoma cells CNE2 in vitro,and the best tumorsuppression activity component was screened out;The purity of the tumor-suppression activity component was testde by RP-HPLC.(3)The effect of the tumor-suppression activity component on the cell morphology of CNE2 was tested by Hoechst33342 staining;the effect of the tumor-suppression activity component on apoptosis rate of CNE2 was detected by FCM;The effect of the tumor-suppression activity component on the migration of CNE2 cells was detected by cell scratch assay.(4)The method of RT-PCR was used to detect the effect of the tumor-suppression activity component on the expression of the h TERT gene of human telomerase reverse transcriptase in CNE2;the method of Western blotting was used to detect the effect of the tumor-suppression activity component on the expression of the h TERT protein of human telomerase reverse transcriptase in CNE2.Results:(1)The 3 components(HI-1,HI-2,HI-3)of Hermetia illucens antibacterial peptides were separated and purified by the Tricine-SDS-PAGE electrophoresis and RPHPLC;The detection of drug sensitive slips method showed that HI-3 had antibacterial activity.HI-3 had antibacterial activity against Staphylococcus aureus,Bacillus subtilis,Escherichia coli and Enterobacter cloacae,the MIC were 80?g/m L,160?g/m L,80?g/m L,80?g/m L respectively.(2)The antibacterial peptide HI-3 can inhibit the proliferation of CNE2 cells in vitro,the inhibition rate can reach 40.56 ± 6.80%,that had a significant difference compared with the HI-1(4.38±0.33%)and HI-2(4.16±0.14%)groups(P<0.05);The purity of active component HI-3 was 96.1% by RP–HPLC.(3)The antibacterial peptide HI-3 could increase the permeability of CNE2 cell membrane;The typical characteristics of apoptosis in CNE2 cells can be observed at 160?g/ m L,the early apoptosis rate was accessibility 27.59±1.14%;Compared with negative control group,it had no obvious alterations for HUV-C cells;The antibacterial peptide HI-3 could decrease the migration of CNE2 cells.The migration rate of HI-3 treatment groups were 24.43±0.47%?61.51±0.04%,and 80±1.46% respectively,at 12 hours,24 hours and 48 hours.Compared with the same period of the negative control group,the migration rate was reduced significantly(P<0.05).(4)The telomerase activity of antibacterial peptide HI-3 treatment groups was significantly lower than that of negative control group,compared with the negative control group,the expression quantity of h TERT gene and h TERT protein were significantly decrease(P<0.05).Conclusion:(1)Small polypeptide with antimicrobial activity(HI-3)can be induced by Staphylococcus aureus acupuncture to produce in the larvae lymph fluid of hermetia illucens.It has a certain inhibitory effect both on Gram positive bacteria and gram negative bacteria;(2)HI-3 could effectively inhibit the proliferation of CNE2 cells and induce its apoptosis and could decreased the migration ability of CNE2 cells;It had little effect on the HUV-C cells;(3)HI-3 could inhibit the expression of telomerase reverse transcriptase h TERT in tumor cells,and decrease the telomerase activity,which can inhibit the proliferation of CNE2 cells and induce the apoptosis of CNE2 cells.