【摘要】 固相法合成Y -box结合蛋白冷休克域部分片段“14肽”(H2 N NGYGFINRNDTKED COOH ,简称ND) ,用不同载体蛋白经戊二醛一步法偶联得到免疫抗原 (BSA ND)和检测抗原 (OVA ND) .用免疫抗原免疫BLAB c小鼠 ,经间接法ELISA检测 ,得到对ND有足够免疫应答的小鼠 .采用常规单克隆抗体制备的方法 ,得到 3株能稳定产生和分泌抗ND单抗的克隆细胞株 ,单抗的亚型均为IgM .WesternBlotting结果显示 :该单抗能识别中华大蟾蜍 (Bufobufogar garizans)Ⅰ、Ⅱ期卵母细胞中的热稳定Y box结合蛋白p5 4 ,并能识别中华大蟾蜍高、低温卵中一种分子量约为 80kD的蛋白 (简称p80 ) .由于p80在低温卵中含量高于高温卵中 ,推测可能是高温卵和低温卵中差异表达的Y box结合蛋白之一 .更多还原

【Abstract】 A segment (14 peptides, H 2N NGYGFINRNDTKED COOH, named as ND) of cold shock domain derived from Y box binding proteins was synthesized by solid phase method and was coupled to different carriers by glutaraldehyde (one step method) to get BSA ND and OVA ND. BSA ND was used to immunize BLAB/c mice. Analyses by indirect ELISA proved that we obtained mice with adequate immunizing response against ND. According to the normal preparation of McAb, three stable monoclonal cell lines were generated finally which can produce and secrete monoclonal antibody against ND. Monoclonal antibodies in the ascites of mice were all typed as IgM. Western Blotting results showed that monoclonal antibody against ND recognized p54, a thermostable Y box binding protein in I、II stage oocytes of Bufo Bufo gagarizans, and could bind to a protein with molecular weight of about 80 kDa (p80) in extract of full grown oocytes derived from Bufo Bufo gagarizans reared at high (28℃) or low (4℃)temperature environments. While the content of p80 in oocytes derived from toad hibernating at low temperature was higher than that at high temperature, which suggested that p80 is probably one of the Y box binding proteins differentially expressed in oocytes and/or sensitively induced by temperature.更多还原