斯氏狸殖吸虫成虫可溶性抗原蛋白质分析

目的分析和检测斯氏狸殖吸虫成虫可溶性抗原的特异抗原组分,为建立特异、敏感的免疫学诊断方法、研制抗斯氏狸殖吸虫药物和疫苗提供理论基础。方法用聚丙烯酰胺凝胶电泳(SDS-PAGE)、双向电泳(Two-dimension-al gel electrophoresis,2-DE)和免疫印迹(Western blot)等方法,对斯氏狸殖吸虫成虫可溶性抗原进行分析。结果SDS-PAGE分离出22条蛋白带,其中主带8条,分子质量单位为13~64 ku,未见65 ku以上条带。Western blot显示20条显色带,主带6条,分子质量单位分别为13、18、22、28、35和64 ku。2-DE电泳分离出斯氏狸殖吸虫成虫可溶性抗原48个多肽斑点,分子质量单位为14~70 ku,绝大部分分布于pI 3.10~7.14,少数分布于pI 8.78~9.85,在pI 7.14~8.78之间几乎未见多肽斑点。Western blot分析出其中的10个主要多肽斑点能被斯氏狸殖吸虫病患者血清识别,大部分为集中在酸性区域的小分子多肽。结论斯氏狸殖吸虫成虫可溶性抗原经SDS-PAGE电泳分离出的22条蛋白带中,有6条蛋白含量较高,能被斯氏狸殖吸虫病患者血清识别。2-DE电泳分离出的48个多肽斑点中,有10个含量较高,能被斯氏狸殖吸虫病患者血清识别,大部分为偏酸性的小分子多肽。

ANALYSIS OF SOLUBE ANTIGENS OF PAGUMOGONIMUS SKRJABINI

Objective To analyze the soluble antigens of Pagumogonmus skrjabini adult worm and demonstrate the specific antigens. Methods 2 dogs were infected with metacercarias of P.skrjabini collected from crab in Xingwen County of Sichuan.After 3 months infection,the adult worms were isolated from infected dog.The soluble antigens of adult worm were analyzed by SDS-PAGE,two-dimensional gel electrophoresis(2-DE) and western blot. Results Using 10% resolution gel and 5% stacking gel,a total of 22 distinct protein bands of the soluble antigens of adult worm were demonstrated by SDS-PAGE after Coomassie Blue R-250 stain.Eight of them were main bands,with MWs 13-64 ku.By western blot,sera of humans infected by P.skrjabini recognized out 20 protein bands with 6 main bands in them.Two-dimensional gel electrophoresis revealed up to 48 individual polypeptidespots in the soluble antigens of adult worm.The pI and MWs of these spots were 3.10-7.14 and 14-70 ku,respectively.By western blot,sera of humans infected by P.skrjabini recognized 10 main polypeptide spots among them.Conclusion 6 protein bands out of 22 soluble antigen bands of the parasite showed high immunogenicity.2-DE revealed up to 48 individual polypeptide spots in the soluble antigens of adult worm.By western blot,sera of humans infected by P.skrjabini recognized 10 main polypeptide spots among them.