| During the Past twenty years, fish immunology research has become the focus ofpreventive medical research in fish, and obtained great progress. Fish immune systemnot only includes the systemic immune system, but includes the mucosal immunesystem consisted of lymphoid tissue samples and mucus secreted by fish skin, gills,gastrointestinal tract and other mucosal tissues. The immune response of fish isinfluenced by environmental factors, especially temperature significantly. Therefore,study of the temperature effect on the fish immune response and the mucosal immunesystem in-depth will help to understand the rules of pathogen invasion, disease andrehabilitation in fish.In this study, the changes of systemic immunity and mucosal immunity at differenttemperatures were analyzed; skin mucus immunoglobulin (Ig) of flounder was purified,and the monoclonal antibody against mucus Ig was prepared; using this monoclonalantibodies, the relationships among mucus Ig, surface Ig-positive cells and serum Ig inflounder were analyzed. Furthermore, cDNA sequence of poly-immunoglobulinreceptor (pIgR) gene of flounder was cloned, the expression level of the gene indifferent tissues was analyzed by RT-PCR analysis, and the protein was recombinedand its characteristics were analyzed. Furthermore, polyclonal antibody againstflounder recombiant pIgR was developed, and the receptor protein in skin mucus wasidentified, which provided a certain value in studying the function of pIgR. Thefollowings are the details:(1) The effect of temperature on immune response of flounder. Using flowcytometric analysis and indirect enzyme-linked immunosorbent assay (ELISA), thedynamics of the immune response in flounder (Paralichthys olivaceus) reared at9,15,21and26°C, was investigated after intraperitoneal injection with inactivated lymphocystis disease virus (LCDV). The results showed that the percentages of surfaceimmunoglobulin positive (SmIg+) cells in peripheral blood, spleen and kidney andserum antibody levels at21°C increased most prominent, while the changes wereslower at lower temperature (9℃and15℃), and the peakwere lower. The peak at26℃was lower than that at21°C. At the same time, the changes of specific antibodies inthe skin mucus were analyzed by ELISA. The results showed the OD value increasedfastest at21℃, and arrived the peak earliest; while the changes were slower at lowertemperature (9℃and15℃), and the peakwas lower. These results indicatedinactivated LCDV elicited the most powerful immune response when floundermaintained at the optimal temperature (21°C) and fish obtained the most effectiveimmunization, while the response was suppressed at9°C,15°C or26°C.(2) Purification of mucus Ig. The flounder mucus Ig was primarily separated byprecipitation with saturated ammonium sulphate, and then further purified by SephacrylS-300gel filtration chromatography and HiTrap DEAE Sepharose Fast Flow Columnusing protein purification system (AKTA prime, Amersham). The protein content waschecked by SDS-PAGE. The results indicated that mucus Ig of flounder wasspecifically purified by the combination of salting-out, Sephacryl S-300gel filtrationchromatography and DEAE Sepharose chromatography, and the purity was high.According to the SDS-PAGE and native-PAGE, the purified mucus Ig showed apparentmolecular weights of72kDa (heavy chain) and26kDa (light chain), and a totalmolecular weight of798kDa.(3) Production and application of monoclonal antibodies anti-flounder mucus Ig.Purified mucus Ig was used to immunize the Balb/C mice, using monoclonal antibodytechnology, eight of positive hybridomas (1A-M2,1C-M10,1E-M2,1F-M7,3F-M9,3G-M5,8B-M4,8E-M8) were cloned because of secreting high titer antibodies. Inwestern blotting, the eight mAbs specifically reacted to the heavy (H) chain of mucusIg. SmIg+lymphocytes in the peripheral blood, spleen, skin and gills of healthyflounder, were analyzed by flow cytometry using mAb1A-M2and mAb2D8againstserum Ig produced in our laboratory before, and the results revealed that both mAbswere reactive with the SmIg+lymphocytes. The positive reactivity rates for mAb 1A-M2were38.64%in the peripheral blood,23.6%in the spleen,16.56%in the skinand6.26%in the gills, while the positive reactivity rates for mAb2D8were48.89%,33.7%,15%and6.02%, respectively, suggesting mucus Ig was similar, but notidentical, to serum Ig. In western blotting, the mAb1A-M2specifically reacted to theheavy (H) chain of mucus Ig, but not reacted with serum Ig of flounder, whereas mAb2D8against serum Ig could react with the H chain of both mucus and serum Ig,indicating the composition of the mucus and serum Ig H chains was different. Theseresultes generated important mucosal immunological information and gave a valuableinsight into understanding the mucosal immunity in flounder.(4) Cloning and expression of pIgR cDNA sequence of flounder. The full-lengthcDNA of pIgR was cloned using rapid amplification of cDNA ends approaches. Thefull-length cDNA of pIgR was of1384bp, containing an open reading frame (ORF) of1005bp encoding a polypeptide of335amino acids with the predicted molecularweight of37.6kDa. At the same time, the expression of pIgR gene was analyzed bysemi-quantitative RT-PCR in different tissues of flounder. The results showed that theexpression of pIgR was higher in the mucosa-associated tissues, and the expressionwas highest in intestines, liver, gills and skin, then was in spleen and kidney, whilewhich was very low in muscle and stomach, indicating that this gene was closelyrelated to transfer of mucus Ig in flounder. At the same time, the ORF was successfullycloned and expressed in Escherichia coli BL21(DE3). The recombinant proteinshowed the molecular mass of58kDa in SDS-PAGE and displayed binding capabilityto flounder IgM detected by ELISA. Furthermore, using the developed polyclonalantibody against recombinant pIgR, a flounder secretory component–like molecule wasonly detected in the skin mucus, not in the serum. All the results indicated that flounderpIgR probably involved in the pIgs transport and provided insights into the roles of fishpIgR in the mucosal immunity. |
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