【摘要】 ESc 6 15是从猕猴中克隆到的一个在附睾中特异性表达的新基因。为了从蛋白质水平深入研究其在精子成熟中的作用 ,在大肠杆菌中表达了该蛋白质的一条含 310个氨基酸的肽段 ,并用其免疫新西兰大白兔 ,得到了滴度为 10 0 0 0 0的抗血清 ;用Western印迹方法鉴定发现该抗血清可检测到 3ng的抗原量 ;并在大鼠附睾组织抽提液中检测到一种能与该抗血清作用的大小约 6 3kD的蛋白质 ,此蛋白质大小与作者实验室在大鼠中克隆到的此基因的同源蛋白质相同 ;利用该抗体通过免疫组化分析确定了ESc 6 15为分泌蛋白质 ,并能与精子结合 ;该抗血清经抗原吸附后阳性结果消失。该高滴度多克隆抗体的获得为ESc 6 15功能的探索提供了一条途径。更多还原

【Abstract】 ESc-615 is a gene which was specifically expressed in epididymis, newly discovered in our laboratory. To elucidate the function of ESc-615 in sperm maturation and fertilization at the protein level, its C-terminal 310 amino acid residues fragment was expressed inE.coli and used to immunize rabbits to obtain polyclonal antiserum with titer of 100 000. The antisera could detect the antigen as low as 3 ng. A 63 kD protein in rat epididymis protein extracts could be detected in Western blot by using this particular antiserum, in accordance with the size of ESc-615 homologue polypeptide deduced from its cDNA. Immunostaining results showed that ESc-615 was a secreted protein and could bind sperms, which was similar with other carboxylesterases in male reproductive system; the positive staining was eliminated after antigen absorption. All these results showed that this antiserum was not only effective but also highly specific, providing a useful tool in further functional studies of ESc-615.更多还原