小鼠Prx2正义和反义真核表达载体的构建与表达

Construction and Expression of Mouse Prx2 Sense or Antisense Eukaryotic Expression Vectors

目的:构建小鼠Prx2正义及反义真核表达载体pEGFP-Nl-sPrx2、pEGFP-N1-aPrx2并在小鼠卵巢颗粒细胞中表达。方法:应用RT-PCR技术,从小鼠卵巢颗粒细胞提取的总RNA中,获得Prx2基因编码序列的正义及反义片段,克隆至真核表达载体pEGFP-N1中,对重组质粒进行酶切和测序鉴定后,以脂质体介导法转染至体外培养的未成熟小鼠颗粒细胞,通过荧光显微镜观察和Westernblot检测其在颗粒细胞中的表达改变。结果:酶切和测序证明重组真核表达载体pEGFP-N1-sPrx2、pEGFP-Nl-aPrx2构建成功,荧光显微镜观察及Western blot确认目标蛋白在颗粒细胞中表达增强或减弱。结论:成功构建小鼠Prx2基因正义及反义真核表达载体pEGFP-N1-sPrx2、pEGFP-N1-aPrx2并在小鼠卵巢颗粒细胞中表达。

Objective： To construct the sense or antisense mouse Prx2 eukaryotic expression vectors pEGFP-NI-sPrx2 and pEGFP-NI-aPrx2. Methods： Sense and antisense Prx2 full length gene were amplified by RT-PCR from the total RNA of the granulosa cells （GCs） of the mouse ovary, then they were cloned into eukaryotic expression vector pEGFP-N1. After the comformation with PCR, restriction endonuclease digestion and the nucleotide sequencing, the recombinant vectors were transfected into the the GCs which were isolated from the immature mice ovaries transiently through lipofectamine mediation.The expression of Prx2 protein were detected with fluorescent inverted microscope and Western Blot. Results： The sense and antisense Prx2 eukaryotic expression vectors were constructed and verified. The expression of Prx2 protein increased in GCs transfected with pEGFP-NI-sPrx2, while which decreased with pEGFP-NI-aPrx2. Conclusion： The eukaryotic expression vectors pEGFP-NI-sPrx2 and pEGFP-NI-aPrx2 have been constructed successfully.