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为研究虾蟹类甲壳动物血细胞膜表面是否具有相同的抗原决定簇及共同抗原表位的生物学特征,采用制备的抗三疣梭子蟹颗粒血细胞单克隆抗体,通过激光共聚焦扫描显微镜(laser scanning confocal microscopy,LSCM)、免疫印迹(Western blot)和流式细胞术(Flow Cytometry,FCM)等多种方法,测定了7种甲壳类动物(凡纳滨对虾、中国明对虾、刀额新对虾、口虾蛄、中华绒螯蟹、日本板蟹及美洲黄道蟹)颗粒血细胞及透明血细胞与三疣梭子蟹颗粒血细胞单克隆抗体发生特异性结合的抗原表位。LSCM可观察到该株抗三疣梭子蟹颗粒血细胞单克隆抗体与中华绒螯蟹和日本板蟹血细胞交叉反应结果为阳性,分析发现在中华绒螯蟹血淋巴中阳性颗粒血细胞在其颗粒血细胞总数中占76.74%,阳性透明血细胞在其透明血细胞总数中占70.59%,日本板蟹阳性颗粒血细胞在其颗粒血细胞总数中占73.86%,阳性透明血细胞在其透明血细胞总数中占16.67%,其余5种甲壳类动物均为阴性;Western blot测试结果显示该株单克隆抗体仅与中华绒螯蟹血细胞反应,且发生反应的抗原决定簇位于分子量为30 ku的蛋白带上;FCM分析发现该株单克隆抗体与中华绒螯蟹透明血细胞和颗粒血细胞均可发生交叉反应,阳性率分别为57.72%和77.05%,与日本板蟹透明血细胞阳性反应极少,阳性率仅为9.57%,与颗粒血细胞发生阳性反应的阳性率为82.59%。
In order to study whether the membrane surface of shrimp, crab crustacean hemocyte has the same biological determinant and the biological characteristics of the common epitope, the prepared monoclonal antibody against C. portularis granulocyte blood cells was analyzed by laser scanning confocal microscopy Microspheres, LSCM, Western blot and flow cytometry (FCM) were used to detect the expression of seven crustacean species (Litopenaeus vannamei, Penaeus chinensis, Shrimp crab, mitten crab, Japanese crab and American yellow crab) granulocytes and transparent blood cells and Portunus trituberculatus granulocyte monoclonal antibodies specific antigen epitopes. LSCM can be observed that the anti-Portunus trituberculatus granulocyte monoclonal antibodies and mitten crab and Japanese crab blood cells cross-reaction was positive, the analysis found that the hemolymph of mitochondria in the haemolymph in its total number of granular blood cells Accounting for 76.74%, positive transparent blood cells accounted for 70.59% of the total number of transparent blood cells, Japanese crab positive granules blood cells accounted for 73.86% of the total number of granular blood cells, positive transparent blood cells in the total number of transparent blood cells accounted for 16.67%, the remaining five kinds of carapace The results of Western blot showed that the monoclonal antibody of this strain only reacted with the hemocytes of Eriocheir sinensis and the antigenic determinant of the reaction was located on the protein band with a molecular weight of 30 ku. FCM analysis showed that the monoclonal antibody The results showed that there was cross-reaction between transparent hemocytes and granular blood cells of E. sinensis, with positive rates of 57.72% and 77.05%, respectively. The positive rate was only 9.57%, which was positive with granulocyte The positive rate was 82.59%.