摘要
目的纯化葎草花粉变应原并分析其免疫学活性。方法粗制葎草花粉提取液经Sephadex G-75 Sephacryl S-200HR层析柱纯化,分段收集各组份。SDS-PAGE检测各组分蛋白相对分子质量,ELISA抑制实验及免疫印迹实验分析各组分蛋白质对特异性IgG和IgE的抑制率及与患者血清的反应率。结果浓缩粗制葎草花粉提取液经Sephadex G-75层析柱纯化,得到两个峰,第一峰富含蛋白质,第二峰含有大量色素,蛋白含量甚微,弃之;收集第一峰及其谷段洗脱液(P液),经Sephacryl S-200HR层析柱纯化,分离出4个组份,即第一峰(P1)、谷段(V)、第二峰(P2)和末段(E)。电泳结果显示纯化后的葎草花粉含20多种蛋白质条带,相对分子质量区带为5.0×103～97.4×103,P1段主要是相对分子质量为43×103～97.4×103的蛋白质,P2段和V段主要是相对分子质量为5.0×103～43×103的蛋白质,E段主要是相对分子质量为5.0×103以下的蛋白质;ELISA抑制实验结果显示P1、V、P2、E对sIgG抑制率分别为68%、70%、95%、5%,对sIgE抑制率分别为25%、64%、71%、11%;免疫印迹实验结果显示P1、V、P2、E与患者血清sIgG的反应率分别为65.63%、78.13%、87.50%、6.25%,与患者血清sIgE的反应率分别为25.00%、71.88%、84.38%、15.63%。结论草花粉含20多种蛋白质成分,相对分子质量在5×103～43×103之间的蛋白质变应原性和免疫原性均较强,为主要变应原;相对分子质量在43×103～94.7×103的蛋白质免疫原性强而变应原性弱,为次要变应原。
Objective To purify the humulus pollen allergen and study the allergenicity and immunogenicity of it. Methods Crude humulus pollen extracts were purified by gel filtration with Sephadex G-75 and Sephacryl S- 200HR. Various fractions of the allergen protein were collected respectively. The molecular weights of protein were confirmed by SDS-PAGE. The inhibition rate and reaction rate with sIgG and sIgE of patient＇s serum were determined by ELISA inhibition test and western blotting. Results Two peaks were obtained from crude humulus pollen extracts by gel filtration of Sephadex G-75. The first peak contained most of protein while the second peak contained little protein and lots of pigments. So the second peak was thrown away. P solution which contained the first peak and valley fraction were purified by gel filtration of Sephacryl S-200HR and four components that included the 1^st peak, the valley, the 2^nd peak and the end fraction were obtained. The results of electrophoresis demonstrated that purified humulus pollen contained more than 20 kinds of protein with the molecular weights ranged from 5.0 ×10^3 to 97. 4 ×10^3. The fraction of the 1^st peak contained protein with the molecular weights ranged from 43 ×10^3 to 97.4 ×10^3, and the fraction of the valley and the 2^nd peak contained protein with the molecular weights ranged from 5.0×10^3 to 43×10^3. The fraction of the end contained protein with the molecular weights lower than 5.0×10^3. The results of ELISA inhibition test showed that the inhibition rate of the 1^st peak, the valley, the 2^nd peak and the end fraction to sIgG were 68%, 70%, 95%, 5% respectively, and those to sIgE were 25 %, 64 %, 71 %, 11 % respectively. The results of western blotting demonstrated that the reaction rate of the of peak, the valley, the 2^nd peak and the end fraction with sIgG of patients＇ serum were 65. 63%, 78. 13%, 87.50%, 6. 25% respectively, and those with patients~ sIgE were 25. 00%, 71. 88%, 84. 38%, 15. 63% respectively. Conclusion Humulus pollen contained more than 20 kinds of protein. The proteins with molecular weights ranged from 5.0×10^3 to 43 ×10^3 were the major allergen with strong allergenicity and immunogenicity. The proteins with molecular weights ranged from 43 ×10^3 to 97.4 ×10^3 were subordinated allergen with strong immunogenicity and weak allergenicity.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2008年第6期1027-1031,共5页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金(批准号30371336)资助
关键词
葎草花粉
纯化
变应原
变应原性
免疫原性
Humulus pollen Purification Allergen Allergenicity Immunogenicity
