Wistar大鼠Pdia3 cDNA的克隆及原核表达

Molecular Cloning and Prokaryotic Expression of Wistar Rat Pdia3

哺乳动物的精卵融合是一个多分子参与的复杂过程,目前只鉴定出几种影响精卵融合的蛋白因子,对其分子机制还知之甚少。其中位于精子上的Pdia3具有二硫键异构酶活性,可能催化其他精卵融合相关蛋白的二硫键变化而参与精卵膜融合。克隆了Wistar大鼠的Pdia3 cDNA片段,长度为1 620 bp,其中编码区为1 518 bp。测序结果显示,编码区内有两个碱基与GenBank公布的大鼠Pdia3 cDNA不同,分别为第147个密码子GAG(Glu)的第1位碱基和第410个密码子AAG(Lys)的第3位碱基,前者造成氨基酸替换,后者仅为多态性。此外,还在大肠杆菌中成功地表达并纯化了GST-Pdia3融合蛋白,为下一步研究大鼠Pdia3在体外影响精卵融合和与其它蛋白的相互作用奠定了基础。

The process of sperm-egg fusion in mammals is a complex mechanism involving multiple proteins,however,little is known about the molecular mechanism underlying this event,except for some proteins have been identified to affect the fusion efficacy.The Pdia3,which has been detected on the sperm head,has an activity of thioredoxin,and was speculated to participate in this process by catalyzing the disulfide bonds to induce conformational changes in other fusion related proteins.In the present investigation,we cloned Pdia3 cDNA of Wistar rat.The cloned fragment is 1 620 bp in length with a CDS region of 1 518 bp.Compared with the rat Pdia3 cDNA sequence released in NCBI database,our cloned Wistar rat Pdia3 has two base substitutions,one is the first base of the 147 codon,resulting in an amino acid change,and the other is the third base of the 410 codon,resulting in a polymorphism.In addition,we successfully expressed GST-Pdia3 fusion protein in E.coli cells.The purified GST-Pdia3 fusion protein lays the foundation for further examination of the effect of Pdia3 on the gamete fusion efficiency and interaction with other proteins in Wistar rat.