| In this dissertation, Mab against hemocyanin of Fenneropenaeus chinensis wasused as the primary antibody, paraffin method and immunohistochemistry wereemployed to observe distribution of hemocyanin in gill, heart, stomach, intestine,lymphoid organ, ovary and hepatopancreas of F. chinensis. Shrimp F.chinensis wereexperimentally infected with white spot syndrome virus （WSSV） supernatantdilutions at10-2,10-4and10-6, respectively, then the content of hemocyanin weredetermined and total protein content in hemolymph was measured. The main aim ofthis dissertation was to provide theoretical basis for the understanding of hemocyaninduring WSSV infection. This dissertation included the following3parts:1. Production of monoclonal antibody against hemocyanin of F.chinensis and Thedetermination of optimal using dilution of antigen and antibodyHybridoma cell strains of monoclonal antibody against hemocyanin of F.chinensiswas resuscitated, the cultured supernatants was collected, the Mab2H3was analysedand characterized with Western-blotting and the optimal using dilution was found outusing ELISA chessboard titration by respective gradient dilution. The result ofWestern-blotting showed that mAb2H3could react with two protein bands withmolecular masses of73and75kDa, which were identified as the subunits ofhemocyanin. The optimal using dilution of antigen and antibody was10-4and2-2.2. Distrubution of hemocyanin in F.chinensis tissuesUsing this Mab that screened out as the primary antibody, paraffin method andimmunohistochemistry were employed to observe distribution of hemocyanin in gill,heart, stomach, intestine, lymphoid organ, ovary and hepatopancreas of F. chinensis.Immunohistochemistry results showed that positive signal was observed in all ofsampled tissues. Strong positive result was found in blood vessel of gill and lacunaes among myocardial bundles of heart; Whereas positive signals presented in lymphcavities and blood sinus of lymphatic organ, hepatopancreatic tubules and blood sinusin connective tissues of mesenteron; Weak positive result could be observed in bloodsinus among oocyte, blood sinus in connective tissues of the inside stomach circularmuscle. No positive signal was observed in negative control. In conclusion,hemocyanin was mostly from the hemolymph rather than fundamental tissue, andwidely distuibuted in various tissues of F. chinensis, and was found abundant in bloodvessel or lacunae where plenty of hemolymph moved through.3．Variations of hemocyanin in shrimp F.chinensis with white spot syndromevirus （WSSV） infectionThe gill tissue （1g） from heavily WSSV infected crayfish （Procambarus clarkii）was homogenized in10ml phosphate buffered saline, then the homogenate wascentrifuged and filtered, the final filtrate was used as viral supernatant（VS）. The VSwas diluted to10-2,10-4and10-6with PBS respectively, shrimps were injected to themuscle with different dilutions of VS. Then survival curve was drawn, accumulativesurvival curve proved that those three VS dilutions could not only insure successfulchallenge to shrimps, but also remain enough survival individuals for sampling, theywere therefore used as infection concentrations in hemocyanin variation assay.Shrimp F.chinensis were experimentally infected with WSSV supernatantdilutions at10-2,10-4and10-6, respectively, After injection, shrimps were collectedrandomly at0,6, l2,24,36,48and72h PI.Gills from sampled shrimps were used for DNA extraction and two-step PCR.Shrimps were firstly detected as WSSV positive at12h PI in10-2VS group in bothstep PCRs, while in10-4VS group, WSSV positive signal occurred at12h PI insecond-step PCR, and in10-6VS group, WSSV positive signal occurred at24h PI insecond-step PCR, after that, the shrimps were all WSSV positive till the end ofinfection. Shrimps in control group were WSSV negative in the whole experiment.The haemolymph from each sampled shrimp was withdrawn for ELISA. Theresults showed in10-2group, hemocyanin content increased significantly at6h（OD405nm=0.74±0.036, ANOVA, p<0.05） and mounted to the peak at12h PI （OD405nm=0.77±0.047, ANOVA, p<0.05）, and decreased close to the control levelat24h PI and significantly decreased till72h PI （OD405nm=0.18±0.046, ANOVA,p<0.05）, however, in both10-4and10-6groups, hemocyanin content had nosignificant change at0h,6h,12h,24h PI, while decreased significantly at36h,48htill the nadir at72h PI (10-4: OD405nm=0.23±0.053;10-6: OD405nm=0.22±0.050,ANOVA, p<0.05).Total protein content in hemolymph was measured by Coomassie brilliant bluemethod, results showed that total protein content of hemolymph in10-2,10-4and10-6groups showed significantly higher than that in control group at6h PI and mounted tothe peak at12h PI (10-2:108.18±1.36mg/ml;10-4:103.49±1.33mg/ml;10-6:96.94±1.06mg/ml, ANOVA, p<0.05), and significantly lower than that in control group at72h PI (10-2:86.95±0.52mg/ml;10-4:89.27±0.76mg/ml;10-6:85.98±0.71mg/ml,ANOVA, p<0.05).Among three VS infection groups, hemocyanin contents significantly increased in10-2group but had no variations in both10-4and10-6VS groups before24h PI, thenafter24h PI, hemocyanin contents in all three VS groups significantly decreased asWSSV infection progressed. It reflected that hemocyanin varied in a dose-dependentmanner at the beginning of WSSV infection, WSSV infectionhas close relationshipwith hemocyanin content. |
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