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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。
C3b receptor rosette experiment was used to investigate the ability of red yeast cells to adhere to different kinds of antigens and to determine the physical and chemical factors that affect the immune adhesion. Three different types of antigens of Vibrio vulnificus, Staphylococcus aureus and Saccharomyces cerevisiae were selected to react with erythrocytes of Japanese flounder under optimal conditions respectively. The results showed that the erythrocytes of Paralichthys olivaceus had immunoadsorption effect on all three antigens, and Vibrio parahaemolyticus, Staphylococcus aureus and Staphylococcus aureus adhered to the erythrocytes of Paralichthys olivaceus at a reaction temperature of 20 ℃ and 0.1 mol / L PBS ~ (++) The C3b receptor rosette rate of S. cerevisiae was (19.00 ± 1.01)%, (6.09 ± 1.36)% and (3.42 ± 0.00)%, respectively. There was significant difference in the immunostaining activity of the three antigens (P <0.05). The erythrocyte membrane protein type 1 complement receptor (CR1) of Paralichthys olivaceus was isolated and purified by Superdex 200 GL high-performance column with AKTA rapid protein liquid chromatography system. The content of CR1 in membrane proteins of erythrocytes was detected by dot-ELISA, Paralichthys olivaceus membrane expressed CR1 molecules with higher homology with higher vertebrates. This experiment proves for the first time that flounder erythrocytes have an important material basis for exerting immune function.