三步沉淀法纯化鸡卵类黏蛋白

Purification of Hen's Egg Ovomucoid by Three-Step Precipitation

鸡卵类黏蛋白(hen’s egg ovomucoid,HOVM)是鸡蛋清中最重要的过敏原蛋白,探讨三步沉淀(一步三氯乙酸沉淀以及两步硫酸铵盐析沉淀)从鸡蛋清中提取并纯化HOVM的工艺条件,并利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳以及高效液相色谱(high performance liquid chromatography,HPLC)技术检测纯化效果。结果表明:用等体积去离子水稀释的鸡卵清液加入质量分数10%三氯乙酸去除大部分杂蛋白后,所得上清液加入硫酸铵至终饱和度50%,盐析沉淀进一步去除非HOVM的杂蛋白,上清液中最后再追加硫酸铵至终饱和度80%,离心所得沉淀再经过透析冻干即为高纯度的HOVM;HPLC(检测波长220 nm)检测所获HOVM的纯度为99.202%,此工艺条件下得到HOVM的提取率约为27.05%。

Hen's egg ovomucoid(HOVM) is the dominant allergenic protein from chicken egg white, and obtaining highpurity HOVM is the prerequisite for understanding its structure and characteristics and reducing its allergenicity. This study presented a three-step precipitation procedure(precipitation with 10% trichloroacetic acid(TCA) followed by two-step fractional precipitation with ammonium sulfate) for purifying HOVM. The purification efficiency was evaluated by SDSPAGE and HPLC. It was found that after most of the protein impurities in the 2-fold diluted egg white were removed by adding 10% TCA, the centrifugal supernatant was added with ammonium sulfate to saturation(50%) for further removal of non-HOVM proteins by salting-out. Finally, additional ammonium sulfate was added to the supernatant fraction until saturation(80%). After centrifugation, the precipitation fraction was dialyzed and freeze-dried to obtain high-purity HOVM product. HPLC analysis(at 220 nm) showed that the purity of the HOVM was 99.202%, with a yield of 27.05%.