Antifungal Activity Of Phosphatidylethanolamine-binding Protein (PEBP) From Silkworm, Bombyx Mori

Bombyx mori belongs to the lepidopteran,which is a typical kind of silking insects.Silkworm spins a cocoon before they pupate.As an important defensive barrier,silkworm cocoons play an extremely important role in the process of external microbes invasion during the pupal period.So which components in silkworm cocoons can resist the invasion of external microorganisms? In 2013,our team used proteomics to analysis protein components from seven kinds of silk.In addition to silk fibroin and sericin,many protease inhibitors,enzymes and unknown functional proteins have been found in silk.In previous studies,it was found that many types of protease inhibitors,which have physiological activity against microbial growth.Protease inhibitors are a very important class of antibacterial proteins.We wonder whether it exists other types of antibacterial proteins in silk.Therefore,the study of antibacterial proteins in silk is of great significance for elucidating the application value of insect defense systems and developing antibacterial silk.In the previous study,we identified a phosphatidylethanolamine-binding protein(PEBP)from silkworm pupa,which was found to be very high abundance in silk.Its homologous protein in Drosophila is thought to function by activating the immune pathway.Overexpression of PEBP in Drosophila could help the larvae to resist bacterial infection and increase the survival rate of larvae.However,until now there is no evidence that Drosophila PEBP can directly inhibit the growth of microorganisms,and the specific mechanism of PEBP involvement in immunity of Drosophila remains unclear.This study is expected to elucidate the biological function of the silkworm PEBP(Bm PEBP)protein,and analysis the specific mechanism by which Bm PEBP protein plays a role in silk.We have studied Bm PEBP from several aspects such as sequence and evolution,expression and distribution characteristics,antibacterial activity and mechanism.The main research results are as follows:1.Basic Information Analysis of Bm PEBP We obtained the amino acid sequence of the Bm PEBP protein from silk DB and searched it in NCBI.The phylogenetic and sequence alignment analysis of PEBP proteins in insects,plants and mammals was carried out by bioinformatics analysis.It was found that the PEBP proteins of insects,plants and mammals were clearly clustered into three branches.The PEBP protein in insects is highly conserved in sequence composition,which is distinct from PEBP protein in plants and mammals.It is mainly reflected in the fact that many insect PEBPs contain signal peptides,while plant and mammalian PEBP proteins do not contain signal peptides,indicating the PEBP protein of insect acquires the ability to secrete extracellularly during evolution.2.Prokaryotic Expression,Purification and Biochemical Analysis of Bm PEBP The primers were designed according to the ORF sequence of Bm PEBP and amplified by PCR.The amplified fragment was ligated into the prokaryotic expression vector PET28 a to obtain the recombinant expression plasmid p ET28a-PEBP.Subsequently,transformating into the expression strain BL21(DE3),after induction expression,a His-tagged soluble protein was obtained in the supernatant at 16 ?.Recombinant Bm PEBP protein with a purity of over 95% was obtained by nickel column affinity chromatography and gel filtration chromatography.The secondary structure of Bm PEBP protein was analyzed by CD.The results showed that Bm PEBP was mainly ?-folded at room temperature.In order to verify the acid-base stability of Bm PEBP,the secondary structure of the recombinant protein Bm PEBP was investigated by CD spectrum in the p H range of 3 to 11.And it was found that the structure of Bm PEBP was also stable in this interval.Temperature stability studies found that when the temperature rised to 60?,the protein began to degrade.3.Study on the Expression Characteristics of Bm PEBP We used fluorescent quantitative PCR to detect the expression pattern of Bm PEBP gene.Tissue expression profiling revealed that the Bm PEBP gene was abundantly expressed in the silk glands on the third day of the fifth instar.Analysis of the expression profile of the period revealed that the Bm PEBP gene was highly expressed on the fifth day of the fifth instar and the seventh day of the fifth instar.Western blot analysis revealed that Bm PEBP protein mainly existed in the middle of silk gland of silkworm.After layering treatment of silkworm pupa,it was found that Bm PEBP protein gradually decreased from the outer layer to the inner layer of silkworm pupa.Immunofluorescence revealed that the Bm PEBP protein was distributed in the outermost layer of a single silk.Therefore,we speculate that Bm PEBP protein is mainly synthesized in the middle silk gland of the fifth instar of silkworm,and secreted into the sericin layer of silk.4.Antibacterial mechanism of Bm PEBP To verify whether the Bm PEBP protein has antibacterial activity,we selected two kinds of bacteria: E.coli and Staphylococcus aureus;four fungi: Candida albicans,Saccharomyces cerevisiae,B.bassiana,Aspergillus fumigatus to study the antibacterial and antibacterial mechanisms of Bm PEBP.Q-PCR and Western blot results showed that Bm PEBP was partially up-regulated in fat and hemolymph after microbial induction.The bacteriostatic kinetic curve indicated that Bm PEBP had a good inhibitory effect on C.albicans,S.cerevisiae,B.bassiana,and A.fumigatus,but had no inhibitory activity against bacteria.The results of circular dichroism showed that PE changed the secondary structure of Bm PEBP,which affected its transformation from ?-sheet to ?-helix.Similarly,binding experiments have also demonstrated that Bm PEBP can specifically bind to PE.Further research and analysis showed that Bm PEBP could be attached to two fungi after incubation with Candida albicans or Saccharomyces cerevisiae.It preliminarily indicated that Bm PEBP was attached to the cells to exert antibacterial action.Because phosphatidylethanolamine is an important component of the fungal cell membrane,we speculate that Bm PEBP may exert antibacterial function by acting on the cell membrane of fungi.