Preliminary Proteomics Analysis Of Hemolymph Of Chinese Shrimp, Fenneropenaeus Chinensis

Chinese shrimp (Fenneropenaeus chinensis) was mainly distributed in Yellow sea and Bosea of China and western coast of Korean Peninsula as an economically and nutritionallyimportant shrimp. Shrimp is the typically mode in crustacean, which's hemolymph is chargedwith many important physiological function and important part in body fluid. Transported intissue by hemolymph, nutriment and hormone keep metabolism and development in body.Hemolymph is temporary metabolize place in shrimp, we could find out the mechanism ofphysiology and pathology efficiently while its protein changed. Shrimp obviously changed inshrimp under the control of some important hemolymph proteins. Some low weight hemolymphpeptides play many important roles in special behavior. The proteomics research of shrimp hasgradually become one important post-genome research focus. In establishing the proteomicresearch platform of shrimp this study, we investigate protein components of shrimp hemolymphby proteomics tools, as will help people understand shrimp on protein level. The works which wedid are showed as follows:1. Study on major high-abundance proteins of F. chinensis hemolymphHemocyanin, a copper containing protein, is the respiratory protein of Crustacea. Here,2major hemocyanin oligomer(hexamers, dodecamers) in the hemolymph of F. chinensis werepurified and studied. Hexamers are composed of3subunits, while dodecamers are composed of4subunits. They possessed3common subunits while dodecamers had a specific subunits.Hexamers and dodecamers occur as major component forms in the hemolymph of F. chinensis,and traces of higher aggregates are aslo found. Hexamers were found to be extremely astableagainst dissociation at high pH, independent of the presence of Ca2+, but dodecamers weredissociated under such conditions.A clottable protein (CP) was purified from the hemolymph of the Chinese shrimp F.chinensis by anion-exchange chromatography and hydrophobic interaction chromatography. Theshrimp CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca2+,suggesting that the clotting reaction is catalyzed by a Ca2+-dependent transglutaminase present inshrimp hemocytes. The molecular mass of the CP was determined to be380kDa under unreducing conditions and190kDa under reducing conditions by SDS-PAGE respectively, andthe protein exists as disulfide-linked homodimers and oligomers. The amino acid sequence at theN-terminus was100%identical to that of the penaeids Litopenaeus vannamei, Farfantepenaeuspaulensis and Penaeus monodon and66%to80%identical to the CPs of ather decapods. ByHomologous Cloning method, a cDNA fragment of CP gene was cloned and sequenced, and itcontained518bp and with deduced amino acid sequences of172residuces. The sequenceanalysis indicated that its shared high identity with CPs gene sequences from other previouslyreported shrimps. RT-PCR analysis revealed that the sub-cuticular and the heart were identifiedas the major tissues that express CP in F. chinensis.2. Establishment of two-dimensional gel electrophoresis system for proteomics analysis of F.chinensis hemolymphThe proteins of shrimp Fenneropenaeus chinensis plasma were separated using immobilizedpH gradient two-dimensional gel electrophoresis (2-DE) after desalting. By optimizing loadingamount, and separation scope of IPG strip, the proteins were successfully extracted from shrimpplasma and were separated by2-DE. After silver staining or Colloidal brilliant blue staining,PDQuest image analysis software was applied to analyze the2-DE images and11of the proteinshighly expressed in shrimp plasma were identified by MALDI-TOF/TOF MS. The resultsrevealed that when a150μg sample, with18cm IPG(pH4～7),after silver staining,2-DEanalysis patterns were obtained；and when a1000μg sample, with18cm IPG(pH4～7), afterColloidal brilliant blue staining,2-DE prepared patterns were obtained. Establishment andoptimization of2-DE patterns laid a foundation for further research of shrimp and othercrustaceans plasma proteomic.To establish the method for removing high-abundance proteins in shrimp plasma proteomicsanalysis, deletion of plasma high-abundance proteins, such as hemocyanins, was practiced byimmobilized metal ion affinity chromatography (IMAC), ultracentrifugation, PEG precipitation.The efficiency of the method was assessed by2-DE. The ultracentrifuge protocol established toremove most hemocyanins was a stable method for removing plasma high-abundance proteins.The proteins spots of2-DE map were more clear and some low-abundance proteins werevisualization after ultracentrifugation. The established methods for high-abundance deletion canbe effectively applied in shrimp and other crustaceans plasma proteomic study.3.2-DE based and ClinProt based differential proteomic analysis of immune-induced F.chinensis hemolymphTo understand immune reponse response of the shrimp to LPS challenged at the protein level,by using two-dimensional polyacrylamide gel electrophesis and PMF, we identified someimportant proteins related with immunity and compared some different protein in this thesis. The protein expressional profiles in hemolymph of LPS challenged and unchallenged shrimp wereexamined by two-dimensional gel electrophoersis (2-DE). Among～300reproducibly detectedprotein spots on each gel,5were up-regulated,10were down-regulated in hemolymph fromimmune-induced shrimp. Mass spectrometry identified15differrntially expressed proteins,including6hemocyanins.The results suggest that these proteins may be involed in innateimmunity, providing new insights for the innate immune mechanisms of the shrimps.To obtain the best experimental results, a preliminary experiment was done to evaluate thereproducibility of this method and to select the optimum experimental materials, e.g. sample type.Then we used ClinProt to assess protein expression profiles and to identify potential serummarkers in immune-induced shrimps. To screen potential specific biomarkers in serum ofimmune-induced shrimps using functional magnetic bead based sample fractiontion combinedwith matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS) technology, The serum samples from10immune-induced and10healthy shrimps wereenriched by the magnetic bead and analyzed by MALDI-TOF MS. The results were analyzed bythe software FlexAnalysis3.0and ClinProTools2.1.103protein peaks were detected at themolecular range of1000～10000Da, among which13ones were significantly different betweenimmune-induced and controls (p<0.05) and all were down regulated in serum ofimmune-induced shrimps. A diagnostic pattern consisting of5protein peaks was established withsensitivity of100%, specificity100%.This study will enhance the understanding of the immune system in the shrimps; enrich theknowledge of the innate immunity in crustacean and have important academic value and goodapplication prospect.