镉对河南华溪蟹卵黄磷蛋白在卵巢中表达含量的影响及ELISA法的建立  

ESTABLISHED OF ELISA METHOD OF VITELLIN FROM FRESHWATER CRAB(SINOPOTAMON HENANENSE) AND EFFECT OF CADMIUM ON VITELLIN ACCUMULATION IN OVARY

作　　者：杨健[1] 刘冬梅[2] 何永吉[1] 汉斯.达姆斯 王兰[1] 

机构地区：[1]山西大学生命科学学院,太原030006 [2]山西医科大学基础医学院,太原030006 [3]高雄医学大学生命科学院,高雄80708

出　　处：《水生生物学报》2015年第2期287-293,共7页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金(No.30870267;No.30970361);山西省回国留学人员科研项目(No.2010015);山西省普通高校特色重点学科建设项目(No.2011-SXDX-SWX-003)资助

摘　　要：为了探究镉对河南华溪蟹(Sinopotamon henanense)的卵子发生的影响,通过凝胶过滤层析法纯化了成熟卵巢中的卵黄磷蛋白(Vitellin,Vn),采用SDS-聚丙烯酰胺凝胶电泳确定了Vn亚基数量及分子量,以纯化的Vn为抗原制备了Vn多克隆抗血清并建立了可靠的Vn含量测定的ELISA方法。采用氯化镉体外亚慢性染毒的方法,运用Vn-ELISA方法检测镉暴露对卵巢组织中Vn含量的影响。实验设对照组和5.8 mg/L镉处理组,处理时间分别为10、15和20d。结果显示,(1)SDS-聚丙烯酰胺凝胶电泳结果表明该蛋白由分子量为102、84和70 k D的3个亚基组成,同时Western-blotting检测表明,抗血清对该蛋白具有较强的免疫特异性;(2)在Vn-ELISA中,Vn抗体最佳稀释倍数为1︰100000,该方法的工作浓度范围为125—2000 ng/m L,定性检测的灵敏度可达到15.62 ng/m L;卵巢中Vn含量测定的结果中各批次间没有显著差异,且平均变异系数分别仅为7.81%,表示该法具有较高的稳定性和重复性;(3)镉引起卵巢组织中Vn含量的显著降低,与镉处理时间具有时间-效应关系,说明了镉暴露可影响河南华溪蟹卵母细胞的成熟并对卵子发生产生抑制作用,进而影响卵巢的正常发育。To explore the effect of cadmium （Cd） on oogenesis of freshwater crab （Sinopotamon henanense）, vitellin （Vn） and the method of Vn-ELISA was studied and established. Vn was purified from mature ovaries of S. henanense by gel filtration chromatography. Using SDS- polyacrylamide gel electrophoresis （SDS-PAGE）, the molecular weight and the quantity of Vn subunit were determined. Based on the purified Vn and Vn anti-serum, the reaction parameters for an enzyme-linked immunosorbent assay （ELISA） was developed. To explore the effect of cadmium on the Vn in the ovaries, freshwater crab S. henanense were treated with 5.8 mg/L Cd for 10, 15, and 20d. The results showed that Vn was com-posed of three subunits （116, 66 and 45 kD）. Western-blotting confirmed the polypeptides had a specific reactivity with mouse polyclonal Vn anti-serum. The optimal dilution rates of Vn anti-serum were shown to be 1︰100000. According to these optimal parameters, the standard linear equation was established for the determination of Vn concentration with a valid range of 125—2000 ng/mL, and qualitative detection can be 15.62 ng/mL. In verification experiments, Vn con-tent in the ovaries showed no significant differences, and the mean coefficients of variation of inter-assay were 7.81%, suggesting that the developed ELISA of this study was precise, stable and repeatable. Moreover, Cd caused a time-dependend down-regulation of Vn level, and showed significant effects of Cd on vitellogenesis, which suggest that Cd slows down oocyte maturation and vitellogenesis in S. henanense.

关 键 词：河南华溪蟹 卵黄磷蛋白 SDS-聚丙烯酰胺凝胶电泳 酶联免疫吸附法 镉 

分 类 号：X174[环境科学与工程—环境科学]