重组质粒pEGFP-N2-Wnt3a的构建和鉴定  

Construction and identification of recombinant plasmid pEGFP-N2-Wnt3a

作　　者：刘亦恒[1] 张寿[1] 吴多庆[1] 曾志谋 黄重[1] 张海英[2] 

机构地区：[1]海口市人民医院骨科中心,海南海口570208 [2]海南医学院人体解剖学教研室,海南海口570208

出　　处：《现代预防医学》2014年第11期2050-2052,共3页Modern Preventive Medicine

基　　金：国家自然科学基金(81100246);海南省自然基金资助(811204);海口市重点科技项目(2011-0135)

摘　　要：目的构建大鼠Wnt3a真核表达重组质粒。方法从大鼠脊髓中提取总RNA,利用逆转录聚合酶链反应(RT-PCR)的方法扩增编码大鼠Wnt3a的基因片段,应用基因重组技术将大鼠Wnt3a基因片段克隆到真核表达载体pEGFP-N2中,经XhoI及EcoRⅠ双酶切和单酶切证实所构建的载体。结果 RT-PCR方法获得大鼠Wnt3a的基因片段,限制性内切酶酶切分析和PCR法鉴定表明为正确重组质粒。结论构建的真核表达重组质粒pEGFP-N2-Wnt3a经过鉴定,结构正确,为进一步转染骨髓基质干细胞(BMSCs)和脊髓损伤(SCI)治疗提供研究基础。Objective To construct recombinant eukaryotic expression plasmid named pEGFP-N2-Wnt3a. Methods Total RNA was extracted from SD Rat＇s spinal cord and the Wnt3a gene was amplified by reverse transcription-polymerase chain reaction （RT-PCR）. By using gene recombination technique, Rat Wnt3a cDNA was inserted into retroviral vector pEGFP-N2. The recombinant plasmid was identified by a pair of specified primers containing the restriction sites of XhoI and EcoR Ⅰ . Results The Wnt3a gene was obtained by RT-PCR, the recombinant plasmid was identified by restriction endonuclease analysis and PCR. Conclusion The recombinant plasmid pEGFP-N2-Wnt3a is constructed successfully, which offers a help for the further research on the expression in bone mesenchymal stem cells and therapy of spinal cord iniure.

关 键 词：Wnt3a基因 pEGFP-N2真核表达载体 脊髓损伤 

分 类 号：R113[医药卫生—公共卫生与预防医学]