| There are many types of microbes in the ocean.In addition to some common terrestrial pathogens identified in marine environment,there are a great variety of specific marine pathogenic fungi,viruses and vibrios.Long exposure of skin or deep tissue wounds to seawater can easily cause infection and severe damage.However,the existing antibiotics are not very effective in the treatment of marine microbial infections.Thus it is urgently needed to search for new antibacterial substances.The physiological structures and metabolites are different between marine and terrestrial organisms,and many bioactive compounds have been isolated from marine creatures,especially from invertebrates.Jellyfish,a representative of macro plankton,is one of the most abundant marine invertebrates.Living in surface seawater,jellyfishes are continuously exposed to many pathogenic bacteria and fungi,which may lead to an increase in the production of immunedefense-associated factors.Until now,there are few reports on the sequence information,expression levels and antimicrobial activities of the immunologic active molecules from jellyfish.Therefore,more sequence data and comprehensive analysis of jellyfish species transcriptome are desired to explore more immunologic active molecules and other bioactive components.Part 1:We performed de novo transcriptome sequencing of the tentacle tissue of C.capillata using Illumina HiSeq? 2000 platform.A systematic bioinformatics strategy was then engaged to make an in-depth and integrated analysis of this transcriptome as well as to identify some other important molecules in C.capillata.A total of 51,304,108 reads were obtained and assembled in 50,536 Unigenes.Among them,21,357 Unigenes had homologues in public databases.Functional annotation of the Unigenes also revealed a general gene expression profile characteristics in the tentacle of C.capillata.We screened out a great number of transcripts encoding proteins similar to several well-known immune defense-associated factors families including protease inhibitors,lectins,BPIs,antimicrobial peptides and HSP.Besides,there are also some transcripts resembling the molecules with potential toxic activities and several degenerative diseases.These results provide a foundation for the future sudy on the functions of these important molecules.Part 2:Several BPIs(bactericidal/permeability-increasing proteins)and a variety of KPIs(Kazal-type proteinase inhibitors),which are believed to play an important role in host immune defenses,have been identified.We first identified and characterized a typical Kazal-type serine protease inhibitor named CcKPI1(C.capillata Kazal-type proteinase inhibitor 1)and a typical bactericidal/permeability-increasing protein named CcBPI1(C.capillata bactericidal/permeability-increasing protein 1)based on the global transcriptome analysis.The physicochemical properties and basic structures of CcKPI1 and CcBPI1 were determined or predicted using a variety of bio-informatics analysis softwares and online tools.CcKPI1 contains an open reading frame of 528 bp encoding a protein of 176 amino acids with a calculated molecular mass of 19.02 kDa,and contains three typical Kazal domains with P1 reactive sites Leu,Arg and Arg,respectively.These results indicated that CcKPI1 might be a new member of Kazal-type proteinase inhibitor family.CcBPI1 contains an open reading frame of 1437 bp encoding a protein of 478 amino acids with a calculated molecular mass of 19.02 kDa,and contains two typical BPI domains,indicating that it might be a new member of bactericidal/permeability-increasing protein family.The CcKPI1 encoding mature protein(without the signal peptide)was amplified and cloned into the expression vector pET24 a,pET28a,pET32 a and pGEX-6P-1,respectively.The recombinant plasmids were then transformed into E.Coli Rosetta(DE3)pLysS strain and E.ColiBL21(DE3)pLysS strain.After induced with IPTG,the recombinant CcKPI1(rCcKPI1)was sucesseful expressed as a GST-fusion protein.The soluble rCcKPI1 recombinant protein was purified using an ?KTA purifier system,the fractions collected at different stages of purification were examined by SDS-PAGE,and a distinct band with a molecular weight of about 43 kDa was detected.The molecular weight corresponded well with the calculated molecule weight(17.43 kDa of mature CcKPI1 protein combined with 26 kDa of GST tag).Western blotting analysis with anti-GST antibody further confirmed that this purified protein was a GST-tagged CcKPI1 fusion protein.The soluble recombinant CcKPI1(rCcKPI1)was then successfully purified.The CcBPI1 encoding mature protein was amplified and cloned into the expression vector pET24a,pET28a,pET32a and pGEX-6P-1.The recombinant plasmid was then transformed into E.Coli Rosetta(DE3)pLysS strain and E.ColiBL21(DE3)pLysS strain.After induced with IPTG,the recombinant CcBPI1(rCcBPI1)was sucessefully expressed as a GST-fusion protein,and a distinct band with a molecular weight of about 79 kDa was detected by SDS-PAGE.The molecular weight corresponded well with the calculated molecule weight(53 kDa of mature CcKPI1 protein combined with 26 kDa of GST tag).Western blotting analysis with anti-GST antibody further confirmed that this purified protein was a GST-tagged CcKPI1 fusion protein.However,the expression level of CcBPI1 was very low that it was difficult to be separated.Therefore,the CcBPI1 encoding mature protein was cloned into the expression vector pPIC9.The recombinant plasmid was then transformed into P.pastoris GS115 strain.Until now,the expression of the recombinant CcBPI1(rCcBPI1)was not yet done.The expression conditions require further grope and optimization.Part 3:Quantitative real-time PCR analysis showed that CcKPI1 and CcBPI1 were expressed in all the tissues tested,including the tentacle,umbrella,gonad and oral arm.The highest expression level of CcKPI1 and CcBPI1 was found in the gonad and oral arm,respectively.A variety of activity assays of the purified recombinant protein were carried out.(1)Five SPIs were used to determine the inhibitory activity of rCcKPI1.The results showed that rCcKPI1 exhibited significant inhibitory activities against elastase,subtilisin A and proteinase K,but not against trypsin or chymotrypsin.The kinetic studies further showed that all the inhibitory effects of rCcKPI1 were competitive,indicating it might be a microbial serine protease inhibitor and could exhibit antimicrobial activity.(2)Microbial binding activity of rCcKPI1 was detected by Western blotting with anti-GST tag antibody.All the microorganisms tested in the microbial binding assay were further used in immunofluorescence staining.The laser scanning confocal microscopy was used to detect rCcKPI1 localized on the microorganisms.As predicted,rCcKPI1 directly bound to various microorganisms,including Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis,Gram-negative bacteria Escherichia coli,marine pathogenic vibrios Vibrio vulnificus,Vibrio cholerae,Vibrio natriegens,Vibrio mimicus,Vibrio alginolyticus and Vibrio parahaemolyticus,and fungi Candida albicans,Candida parapsilokis and Candida glabrata.(3)The ability of inhibiting the growth of rCcKPI1 protein was detected by micro liquid dilution method.The results showed that rCcKPI1 could inhibit the growth of most of the tested microorganisms that it binds to.Conclusions:In this study,we performed de novo transcriptome sequencing of the tentacle tissue of C.capillata,and made an in-depth and integrated analysis of this transcriptome to explore some important molecules in C.capillata.We reported,for the first time,the KPI and BPI in jellyfish,and also demonstrated that CcKPI1 exhibited a marked antimicrobial activity indicating that CcKPI1 act as an antimicrobial factor and might protect the jellyfish C.capillata from the potential pathogens in marine environment.This study may provide scientific foundation for the understanding of the innate defense mechanism of jellyfish,and facilitate the research on antimicrobial agents from the marine natural products. |
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