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小鼠Prx2正义和反义真核表达载体的构建与表达
引用本文:杨书红,吕兴,罗爱月,赖志文,丁婷,韩凌斐,卢运萍,王世宣.小鼠Prx2正义和反义真核表达载体的构建与表达[J].生物磁学,2011(16):3005-3008.
作者姓名:杨书红  吕兴  罗爱月  赖志文  丁婷  韩凌斐  卢运萍  王世宣
作者单位:[1]华中科技大学附属同济医院妇产科,湖北武汉430030 [2]郧阳医学院附属太和医院肝胆外科,湖北十堰442000
基金项目:卫生部公益性行业科研专项项目(200802159);教育部高等学校博士点科研基金(200804870009)国家自然科学基金资助项目(30973148)
摘    要:目的:构建小鼠Prx2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。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衰达载体pEGFP—N1一sPrx2、pEGFP—N1-aPrx2构建成功,荧光显微镜观察及Westernblot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。

关 键 词:Prx2  正义/反义  真核表达载体  颗粒细胞  小鼠

Construction and Expression of Mouse Prx2 Sense or Antisense Eukaryotic Expression Vectors
YANG Shu-hong,LV Xing,LUO Ai-yue,LAI Zhi-wen,DING Ting,HAN Ling-fei,LUYun-ping,WANG Shi-xuan.Construction and Expression of Mouse Prx2 Sense or Antisense Eukaryotic Expression Vectors[J].Biomagnetism,2011(16):3005-3008.
Authors:YANG Shu-hong  LV Xing  LUO Ai-yue  LAI Zhi-wen  DING Ting  HAN Ling-fei  LUYun-ping  WANG Shi-xuan
Institution:1 Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, PR China; 2 Hepatobiliaty Department of Taihe Hospital, Yunyang Medical College, Shiyan , Hubei 442000, China)
Abstract: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.
Keywords:Prx2  Sense/Antisense  Eukaryotic expression vector  Granulosa cells  Mouse
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