| Viral infection induces host immune response. Some factors in host cells playvital roles in the process above, and in general, their expression levels are up-ordown-regulated. White spot syndrome virus (WSSV) disease is one of the mostdevastating diseases in shrimp aquaculture, and Chinese shrimp (Fenneropenaeuschinensis) is its susceptible host. In order to identify the key proteins which playimportant roles in responses of shrimp to WSSV infection, proteomic approach wasused in the present work to identify differentially expressed proteins in F. chinensishemocytes. Moreover, further analysis was done to investigate the characters andfunctions of two identified proteins, SUMO and protein phosphatase, in WSSVinfection. The results provided important data on the molecular mechanism of shrimpanti-WSSV responses. The details of research contents and results are as follows:(1) Proteomic analysis of differentially expressed proteins in F. chinensishemocytes upon WSSV infection. Two-dimensional (2D) gel electrophoresis wasapplied to separate the hemocytes samples collected form WSSV-infected and controlgroups, and PDQuest software was used to analysis the2D gels. The results showedthat a2D image of total protein from F. chinensis hemocytes was obtained, andapproximately580protein spots were detected in the image. At24h post infection(hpi),26protein spots were significantly up-regulated (P<0.05), and19spots weresignificantly down-regulated. By mass spectrometry, small ubiquitin-like modifier(SUMO)1, cytosolic MnSOD, triosephosphate isomerase, tubulin alpha-1chain,microtubule-actin cross-linking factor1, nuclear receptor E75protein, vacuolar ATPsynthase subunit B L form, inositol1,4,5-trisphosphate receptor, arginine kinase, etc.,amounting to33differentially modulated proteins were identified successfully.According to Gene Ontology annotation, the identified proteins were classified into nine categories, consisting of immune related proteins, stimulus response proteins,proteins involved in glucose metabolic process, cytoskeleton proteins, DNA or proteinbinding proteins, proteins involved in steroid hormone mediated signal pathway, ATPsynthases, proteins involved in transmembrane transport and ungrouped proteins.Moreover, the expression profiles of three up-regulated proteins (cytosolic MnSOD,heat shock protein70and arginine kinase) and one down-regulated protein(prophenoloxidase) were further analyzed by real-time RT-PCR at the transcriptionlevel after WSSV infection. The results were consistent with the proteomic data.(2) Cloning and expression analysis of SUMO and SUMO-conjugating enzymeE2UBC9from F. chinensis. SUMO was chosen from the identified proteins, and thefurther analysis was performed. Full length cDNAs of SUMO (FcSUMO) and UBC9(FcUBC9) were cloned from F. chinensis using rapid amplification of cDNA endsapproach (RACE), and they were1128bp containing an open reading franme (ORF)of282bp and1170bp containing an ORF of483bp respectively. The ORF ofFcSUMO encoded a93amino acids peptide with the predicted molecular weight(M.W) of10.6kDa, and the UBC9ORF encoded a160amino acids peptide with thepredicted M.W of18.4kDa. Homology comparisons indicated that the amino acidsequences of the two genes were both highly conserved. By quantitative real-timeRT-PCR, FcSUMO and FcUBC9mRNAs were observed in all the eight tissues ofhealthy F. chinensi, with the highest transcription levels in hemocytes and gonad, andthe two genes were both up-regulated after WSSV infection，while the fold changes ofFcSUMO and FcUBC9transcripts in hemocytes were both higher than that in gonad..(3) Prokaryotic expression of FcSUMO ORF and FcUBC9ORF and preparationand application of anti-SUMO or anti-UBC9polyclonal antibodies. The recombinantplasmids, pET-28a-SUMO and pET-28a-UBC9, were constructed successfully inEscherichia coli. After induced expression, the recombinant proteins of FcSUMO(rSUMO) and FcUBC9(rUBC9), with M.W of19.7kDa and25.6kDa respectively,were obtained. Subsequently, the two recombinant proteins were purified andsubjected to the polyclonal antibody (PAb) production, respectively. Characterizationsof SUMO and UBC9in F. chinensis hemocytes were performed using the PAbs. By western blotting, a13.5kDa protein and a18.7kDa protein in hemocytes wererecognized by the PAb against SUMO or UBC9respectively. Byimmunofluorescence assay, the positive signals of SUMO were observed in nucleusand cytoplasm of hemocytes, and the positive signals of UBC9were mainly detectedin nucleus.(4) RNA interference and WSSV infection. Double-stranded RNA (dsRNA)corresponding to FcSUMO and FcUBC9sequences were generated by in vitrotranscription. To investigate the effects of suppression of the FcSUMO and FcUBC9transcripts on WSSV infection, shrimp were grouped and injected with dsRNAs. Theresults showed that the number of WSSV copies and the viral gene expressions wereinhibited by knockdown of either SUMO or UBC9, and the mortalities of shrimp werealso reduced.(5) Cloning and prokaryotic expression of protein phosphatase1catalytic subunitbeta isoform (PP1β) in F. chinensis. PP1β of F. chinensis (FcPP1β) was cloned andsequenced by RACE technology. The full-length cDNA sequence of FcPP1β was1214bp, and contained an ORF of987bp that encoded for a polypeptide of328amino acids with the predicted M.W37.6kDa. Homology comparisons indicated thatthe amino acid sequence of FcPP1β was highly conserved. By quantitative real-timeRT-PCR, PP1β mRNA was observed in all the eight tissues of healthy F. chinensis,with the highest transcription levels in gonad and hemocytes. Moreover, the gene inthe above two tissues was up-regulated after WSSV infection, with the peak value at12hpi，while the fold changes of FcPP1β transcripts in hemocytes were higher thanthat in gonad. Moreover, the recombinant plasmid of FcPP1β ORF, pET-28a-PP1β,was constructed successfully in E. coli. After induced expression, the recombinantprotein with M.W of41kDa was obtained, and then the protein was purified. |
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