Wistar大鼠CD9的cDNA克隆、原核表达及生殖腺中的检测

Molecular Cloning,Prokaryotic Expression and Identification of CD9 in Gonads of the Wistar Rat

为了用Wistar大鼠研究CD9对精卵融合的影响和与其它精卵融合相关蛋白的作用,克隆了Wistar大鼠的CD9cDNA。测序结果显示,Wistar大鼠的CD9 cDNA编码区与GenBank中发布的SD(Sprague-Dawley)大鼠相同,但在3'非翻译区多一个T。用Western blotting方法检测Wistar大鼠睾丸和卵巢总蛋白发现睾丸和卵巢里均表达内源性CD9蛋白,分子量相同。此外,在大肠杆菌中表达了GST-CD9融合蛋白,并用GST标签纯化CD9蛋白,为体外研究CD9与其它精卵融合相关蛋白的作用提供参考。

CD9 is essential to mammalian sperm-egg fusion.For studying on the effect of CD9 on gamete fusion and the interaction of CD9 with other sperm-egg fusion related proteins in the Wistar rat,we cloned CD9 cDNA of the Wistar rat.The cDNA sequence of the Wistar rat CD9 is identical to the SD（Sprague-Dawley）rat released in GenBank in the coding region,but with an extra T in the 3＇ UTR.Western blotting analysis of the protein extracts of the gonads of the Wistar rat shows that endogenous CD9 is synthesized with the same molecular weight in both testis and ovary.In addition,we also expressed GST-CD9 fusion protein in E.coli BL21（DE3）and purified the Wistar rat CD9 protein using the GST tag.These results lay the foundation for examining the interaction of CD9 with other sperm-egg fusion related proteins in vitro.