瓦氏黄颡鱼卵黄脂磷蛋白(Lv)的纯化、性质鉴定及其抗血清的研制

Purification and characterization identification of lipovitellin from Pelteobagrus vachelli and preparation of anti-serum

采用凝胶过滤和离子交换两种层析技术,从瓦氏黄颡鱼Ⅳ期卵巢粗提液中分离、纯化出卵黄脂磷蛋白(Lv),采用糖、磷、脂蛋白染色技术验证分离、纯化的蛋白为Lv,该Lv在非变性条件下分子量约为230ku,在SDS变性条件下分子量约为106ku。纯化的瓦氏黄颡鱼Lv经检测显示含有类胡萝卜素,但没有二硫键,对热相对稳定。利用纯化的瓦氏黄颡鱼Lv,制备了兔抗瓦氏黄颡鱼Lv多克隆抗血清。通过双向免疫扩散法测得Lv抗血清的效价为1∶32,还发现瓦氏黄颡鱼卵黄蛋白原(Vtg)和Lv之间有明显的免疫交叉反应性,说明两者具有相同的免疫原性;Western-blotting检测显示抗血清的特异性较好,并能特异性地识别Vtg。

Lipovitellin （Lv） is one of the major proteolytic products of vitellogenin （Vtg） after it is taken up by growing ovary in the manner of receptor-mediated endocytosis, which provides nutrient storage for the developing embryos and larvae. In this paper, we purified the Lv from ovarian extract of the Pelteobagrus vachelli in stage 1V by two steps of chromatography elution method. The first step of purification was to use Sephacryl S-300 column, and 3 etution peaks were showed. We are suspicious that the 3th elution peak contained Lv through analysing the Native-PAGE chart and elution curve of stage Ⅳ ovarian extract. The second step of purification was to use DEAE-cellulose column in ion-exchange chromatography elution, and there were 2 elution peaks. We found that the 2th elution peaks（ in 0.2 mol/L NaCl gradient） contained single high concentration of protein by Native-PAGE analysing of the sample. The Lv characterized as a phosphorlipoglycoprotein by Native-PAGE and stained of gels for carbohydrates with periodic acid-Schiff＇ s reagent, for phosphorus with Rhodamine B, and for lipids with Sudan black B. The molecular weight of Lv is about 230 ku detected by Native-PAGE. The Lv broke into 2 same subunits in Sodium dodecyl sulphate- PAGE,each with a molecular weight of 106 ku. In 190 - 1 000 nm continuous spectrum scanning, the purified Lv of P. vachelli obviously showed absorb peak in 273 nm, and it demonstrated that the Lv contained carotenoid. Native-PAGE analysis of the purified Lv of P. vachelli show that there is no disulfide bond. It is relatively stabilized by heated treatment. We prepared the rabbit polyclonal antiserum against P. vacheUi Lv by using purified Lv. The titre for Lv polyclonal antisera was 1 ： 32 in double immunodiffusion assay detection. It was showed that the purified Vtg which was induced by E2 in male P. vachelli serum could react with the rabbit antisera against P. vacheUi Lv and the purified Lv could react with the rabbit antisera against P. vachelli Vtg and a single immunoprecipitin line was formed. The plasma from the control male P. vachelli have no reaction. The normal existence of the female-specific reactivity for P. vachelli Lv can be confirmed. The test also demonstrated that both purified Vtg and stage IVovarian extract from the P. vachelli can cross-react with the rabbit Lv polyclonal antiserum by the analyses of Native-PAGE and Western-blotting. We are sure that the rabbit Lv polyclonal antiserum has preferable specificity with Vtg and Lv.