Gene Identification,preparation And Functional Exploration Of Defensins From Oysters And Anopheles

Defensins are the largest family of antimicrobial peptides(AMPs)and are considered to form part of the innate immunity in host defense system.They are produced in vertebrates,invertebrates,and even plants and fungi,widely found in nature.They are usually short cationic peptides with 2-4 disulfide bonds and 34-54amino acid residues.Oysters live in the intertidal zone or subtidal zone of the ocean,which are always in contact with the abundant source of pathogens in the ocean.Mosquitoes breed in sewage,prefer humid and dark environment,and carry a variety of mosquito-borne viruses,plasmodium,filaria and other pathogens.In the face of ubiquitous pathogenic microorganisms,oysters and mosquitoes do not have perfect specific immune responses,but their innate immunity is amazing.As the effector of the innate immune response,defensins have a variety of biological activities such as broad-spectrum anti-bacterium,anti-fungus,anti-virus,anti-plasmodium and so on,providing the possibility for oysters and mosquitoes to resist the invasion of foreign pathogenic microorganisms.As one type of insects from Arthropods,mosquito has many unique biological characteristics.For example,mosquitoes carry a variety of mosquito-borne viruses(Zika virus,Dengue virus and Japanese encephalitis virus),plasmodium parasites,filariasis and other pathogens,and spread these pathogens through bites,resulting in widespread epidemics of related diseases.Another characteristic of mosquitoes is the significant itch pathology when they bite humans and other mammals.It is well known that the salivary glands secreted by mosquitoes not only contain pathogens,but also have abundant polypeptide components,among which defensin is one class of the important peptide components.However,it is not still known whether the defensins secreted by mosquitoes'salivary glands are involved in virus transmission and causing itch.First,three defensin genes from the Pacific oyster Magallana gigas were identified.We started to search for defensin genes in the Pacific oyster genome,found23 oyster defensins containing four pairs of disulfide bonds in the literature,and then download their CDS sequences and the entire Pacific oyster genome on the NCBI website.After local blast alignment,three unique defensin sequences of Magallana gigas,Mgdef-1,Mgdef-2 and Mgdef-3 were obtained.Their gene sequences and c DNA sequences were successfully cloned and sequenced by molecular biology techniques,and their sequence and structure biological analysis were performed,which confirmed the three defensins in Magallana gigas at the gene level and the transcriptional level.Second,the recombinant peptide rMgdef-1 and anophele defensins Asdef-1 and Agdef-2 were prepared.rMgdef-1 was obtained by prokaryotic expression,affinity chromatography,enzyme digestion and reversed-phase high performance liquid chromatograph(RP-HPLC).The peptide with 40 amino acid residues formed 4 pairs of disulfide bonds.Its molecular weight was identified to be 4291.35 Da,which was in line with the expected molecualr weight.The reduced-type linear peptides of Anopheles stephensi defensin and Anopheles gambiae defensin were obtained by chemical synthesis method.After dissolution,it was oxidized in a Tris-HCl buffer system to form 3 pairs of intramolecular disulfide bonds.The oxidized-type peptide was separated and purified by semipreparative RP-HPLC.The molecular weights of the oxidized-type peptides were identified by mass spectrometry to be 4194.10 Da and 4682.37 Da,indicating that Ox-Asdef-1 and Ox-Agdef-2 were prepared sucessfully.Finally,the bilogical functions of three defensins rMgdef-1,Ox-Asdef-1 and Ox-Agdef-2 were preliminarily investigated.The antibacterial activities of the three defensins against two gram-positive bacteria and two gram-negative bacteria were determined by the inhibition zone test and the minimum inhibitory concentration test.The results showed that rMgdef-1 and Ox-Asdef-1 have inhibitory effects on the gram-positive bacteria S.aureus AB94004.Ox-Asdef-1 also has good antibacterial activity against the gram-negative bacteria E.coli ATCC25922,while Ox-Agdef-2fails to show antibacterial activity against the above four standard strains.As mosquito bites can spread Zika virus(ZIKV),the effects of Ox-Asdef-1 and Ox-Agdef-2 on ZIKV were detected by virus infection cell assay.The results showed that Ox-Asdef-1 and Ox-Agdef-2 had no anti-ZIKV effect,which was different from the antiviral property of many defensins.Since mosquito bites mostly induce the itching sensation in the body,we used a mouse itch model combined with Ca²⁺fluorescence microscopic imaging technique to show that two anopheles defensins,Ox-Asdef-1 and Ox-Agdef-2,can both induce the scratching behavior in mice.We also found that the molecular mechanism of pruritus was induced by the activation of the Mrgpr C11/Mrgpr X1-TRPV1 signal axis in the peripheral nervous system.In conclusion,three defensins were identified from Magallana gigas,and one of them was found to have antibacterial activity,which enriched the understanding of antimicrobial peptides from marine animals.Ox-Asdef-1 and Ox-Agdef-2 were prepared by oxidative refolding.Different from other defensins,the two anopheles defensins have no anti-ZIKV effect,and may be positively correlated with the transmission of ZIKV through mosquito bites.Ox-Asdef-1 and Ox-Agdef-2 were found to induce itchy sensation by activating Mrgpr C11/X1-TRPV1 signal axis,which may partially explain the itch pathological phenomenon of mosquito bites,and provide a new strategy for the prevention and treatment of mosquito bite-related itch.