缢蛏过敏原的分离、鉴定与纯化

Purification and identification of allergens in Razor clam

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110kD、55kD、42kD、38kD、35kD和28kD,其中以35kD处最为富集.经Western-Blotting鉴定,58kD蛋白为缢蛏的主要过敏原,38kD、28kD和12kD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,58kD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,58kD的主要过敏原位于反相纯化的第2峰.

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.