Preliminary Analysis Of Biological Activity Based On Apis Mellifera Ligustica Spinola Crude Venom

Apis mellifera Ligustica Spinola is a kind of honeybees,which attaches Animal kingdom Arthropoda Phylum Arthopoda,hexapoda,Hymenoptera,Apoidea,Apidae on the taxonomy.Apis mellifera Ligustica Spinola is widely distributed in the world and largely utilized in various yields.Bee venom is transparent liquid which contains various active substances,including Melittin,Apamin and Hyaluronidases,etc.In this study,we investigated venom's material composition and biochemical properties by collecting the venom from Apis mellifera Ligustica Spinola and testing the toxic effect on the leukemia cell K562 at the crude venom level.It was found that the IC50 of K562 cells was 5.49?g/mL(n=5).Afterwards,we evaluated changes in the morphology of leukemia cell K562 by using inverted microscope and found that 5?g/mL of crude venom made leukemia cells membrane damaged a bit and irregular in shape,the cells were all dead with a broken membrane when treated with 10 ?g/mL BV crude venom.To verify whether there are any components acting on the ion channels,the reversed-phase high performance liquid chromatography(HPLC)was used to separate and purify proteinous components.We got 20 main peaks and pooled them,respectively.Then they were dehydrated by vacuum freeze-drying.The electrophoretic detection of each component was carried out.It was found that the peak with retention time of 8min lead to complete inhibition of the sodium channel currents expressed in ND7/23 cells.It has been reported that several kinds of voltage-gated sodium channels subtypes were expressed in ND7/23 cell lines,thereafter,we demonstrate that the selectivity of the toxin.The Nav1.3,Nav1.4,Nav1.5 and Nav1.7 were detected by the heterologous expression of HEK293T cells and Nav1.8 was expressed in ND7/23cell.Our findings suggested that the venom inhibited the currents of Nav1.3 and Nav1.4 completely,and 60%currents of Nav1.5,52%currents Nav1.7 and 93%currents Nav1.8,we concluded that Apis mellifera toxins has a certain selectivity on the sodium ion channels.It has been shown previously that Lycosin-I was an antimicrobial peptide with excellent inhibitory effect on various clinical pathogenic strains.Weather the Lycosin-I could improve the antibacterial activity by chemical modification,the hypothesis was tested by coupling a glucose molecule at the amino end(called Glu-Lycosin-I)and then assessed the antimicrobial activity between wild-type Lycosin-I and Glu-Lycosin-I,the results indicated that the antibacterial activity of modified Glu-Lycosin-I was weaker compared to wild-type on several clinical pathogens,which means the current modification method does not work well.Then,we will try to combine different molecules with Lycosin-I to make it a better antibacterial peptide.