缢蛏过敏原的分离、鉴定与纯化-《深圳大学学报理工版》

文章信息/Info

Author(s):: LI Li; LI Qi-yuan; LIU Zhi-gang; and YU Xiao; College of Life Science，Shenzhen University，Shenzhen 518060，P．R．China

Keywords:: Razor clam; allergen; SDS-PAGE; Western-blotting; high pressure liquid chromatography

摘要:: 以磷酸盐缓冲液提取蛋白，通过聚丙烯酰胺凝胶电泳(SDS-PAGE)对其抗原成分进行分析，选用缢蛏过敏患者的阳性血清进行免疫印迹，鉴定出主要与次要过敏原．用高压液相色谱(HPLC)纯化主要过敏原蛋白，鉴定其免疫活性，结果表明，缢蛏粗提液SDS-PAGE显示有18条蛋白条带，含量高的蛋白有6条，其分子量分别为110 kD、55 kD、42 kD、38 kD、35 kD和28 kD，其中以35 kD处最为富集．经Western-Blotting鉴定，58kD蛋白为缢蛏的主要过敏原，38 kD、28 kD和12 kD蛋白为缢蛏的次要过敏原．HPLC体积排阻纯化后得9个峰，以SDS-PAGE检测，58 kD的蛋白位于第4峰，其中有一条明显的蛋白条带．将该蛋白条带用HPLC反相纯化，得到两个峰．经Western-Blotting鉴定，58 kD的主要过敏原位于反相纯化的第2峰．

Abstract:: To purify and characterize the allergens of Razor clam(Sinonovacula constricta)，the protein was extracted with phosphate buffer solution．The allergens in Razor clam were characterized with 9 allergic patients’ serum by Western-blotting after SDS-PAGE．To purify the primary allergen，HPLC method was employed．Finally immune activeness of the primary allergen was characterized by Western-blotting．The results indicate that，in the crude extracts，there are eighteen protein bands visible on the SDS-PAGE gel．The most abundant six bands are 110 kD，58 kD，42kD，38 kD，35 kD and 28 kD proteins，respectively．We identified the primary allergen whose molecular mass is 58 kD by Western-blotting，and the minor allergens whose molecular mass is 38 kD，28 kD and 12 kD，respectively．Upon HPLC-exclusion chromatogramphy of the crude extract，we obtained 9 peaks．After SDS-PAGE，the 58 kD protein was found in the forth peak．The protein in the forth peak was further purified by reversed phase high performance liquid chromatography．We obtained 2 peaks from the second purification．After Western-blotting ，we found that the 58 kD primary allergen was in the second peak．Thus，we obtained the pure primary allergen in Razor clam．