黑龙江茴鱼卵黄脂磷蛋白分离纯化及抗血清的研制

Isolation and Purification of Lipovitellin from Thymallus arcticus grubei and Preparation of Anti-serum

采用SephadexG-200凝胶过滤层析法,对黑龙江茴鱼卵黄蛋白粗提液进行分离纯化,层析洗脱共得到3个蛋白峰。用聚丙烯凝胶电泳、免疫扩散等方法研究表明:黑龙江茴鱼卵黄蛋白(Yolk protein)含有分子量分别为567.2,486.3,157.5 kDa的3种蛋白,由分子量分别为80.7,68.7,46.8,39,30,20.9,16.7,14.9,14.1 kDa的9个蛋白亚基组成;峰2蛋白为卵黄脂磷蛋白(Lipovitellin,Lv),分子量为486.3 kDa,由分子量为80.7,30,14.9,14.1 kDa的4个蛋白亚基组成;纯化的黑龙江茴鱼Lv免疫新西兰大白兔获得兔源多克隆抗血清,用免疫扩散的方法测定Lv抗血清效价为1∶64;免疫双扩散检测表明,抗Lv血清表现出明显的雌性特异性。

The yolk protein is the main nutrients during embryonic development. The quantity and quality of yolk is crucial for early larval growth. We have purified the egg curde extract of Amur grayling by Sephadex G-200. The results showed there were three protein peaks 1,2 and 3 by gel chromatography at 4℃ 0.2 mmol/L PMSF and three protein peaks were relatively symmetric. Then protein peak 2 was purified by more than one time Sephadex G- 200 eluting. The properties of yolk protein were analyzed by the methods of polyacrylamide gel electrophoresis and immuno diffusion. Based on the 7.5 % Native-PAGE staining data, three brands were detected with molecular weights of 567.2,486.3,157.5 kDa. Based on the 12% SDS-PAGE staining data,nine brands were detected with molecular weights of 80.7,68.7,46.8,39,30,20.9,16.7,14.9,14.1 kDa. Protein peak 2 was lipovitellin and molecular weight was 486.3 kDa. Lipovitellin was composed of three subunits with molecular weights of 80.7,30,14.9,14.1 kDa, respectively. Polyclonal antibodies against the purified Lv from Amur grayling were produced in the New Zeal- and rabbit. Double immunodiffusion showed that the determination of the titre for anti-Lv serum was 1 ： 64. Double immunodiffusion showed that anti-Lv serum have sex-specificity.