牙鲆红细胞对不同抗原的免疫黏附及Ⅰ型补体受体的检测

Paralichthys olivaceus erythrocytes' immune adherence to different antigens and detection of their complement receptor Ⅰ

采用C3b受体花环实验探究了牙鲆红细胞对不同种类抗原的免疫黏附能力,确定了影响免疫黏附作用的理化因子。实验选取创伤弧菌、金黄色葡萄球菌和啤酒酵母3种不同类别的抗原分别与牙鲆红细胞在优化条件下反应。结果表明,牙鲆红细胞对3种抗原均有免疫黏附作用,在20℃、0.1mol/L PBS^(++)缓冲液的反应条件下,牙鲆红细胞黏附创伤弧菌、金黄色葡萄球菌和啤酒酵母的C3b受体花环率分别为(19.00±1.01)%、(6.09±1.36)%和(3.42±0.00)%,对3种抗原的免疫黏附活性差异显著(P<0.05)。利用AKTA快速蛋白液相层析系统的Superdex 200 GL型高效柱分离纯化牙鲆红细胞膜蛋白Ⅰ型补体受体(CR1),通过dot-ELISA法检测发现红细胞膜蛋白组分中含有CR1成分,表明牙鲆红细胞膜上表达了与高等脊椎动物有较高同源性的CR1分子。此实验首次证明了牙鲆红细胞具有发挥免疫功能的重要物质基础。

The C3b receptor rosette technique was employed to investigate the erythrocytes immune adherence activity of lefteyed flounder （ Paralichthys olivaceus ） to different antigens under the optimized immune adherence conditions. Three dif- ferent kinds of antigens （gram-negative bacteria, gram-positive bacteria and eukaryote） were chosen to incubate with ery- throcytes in the 0.1 mol/L PBS＋＋ buffer at 20℃for 30 min. The results indicated that the flounder erythrocytes were able to adhere to all the three kinds of antigens with the C3b receptor rosette rates for Vibrio vulnificus, Staphylococcus aureus and Saccharomyces cerevisiae being （19.00± 1.01）%, （6.09± 1.36）% and （3.42 ± 0.00）% respectively. The external structures of the antigens were inferred to be responsible for the significant difference of rosette rates. Fur- thermore, erythrocyte membrane proteins were extracted and purified by the Superdex 200 FPLC gel chromatography to prepare the complement receptor I （CR1） component. The Dot-ELISA assay confirmed there was the CR1 component in erythrocyte membrane proteins and the CR1 molecule homologous to higher vertebrate was expressed on erythrocytes of left-eyed flounder. Erythrocytes have been proved to possess key immune molecules and play an important role in the lefteyed flounder immune system in the study.