医学pcDNA3.1preS1GFP重组粒构建及其小鼠精子表达.docVIP

pcDNA3.1preS1GFP重组质粒构建及其小鼠精子表达 作者：李茜 王培林 王亮 张喆 郑征 【摘要】 目的 构建绿色荧光蛋白(GFP)与乙型肝炎病毒(HBV)preS1基因的重组真核表达载体并观察其在小鼠精子内的表达。方法 采用PCR方法,以HBV全基因组质粒为模板扩增HBV preS1基因片段,利用DNA重组技术将其定向插入到含有GFP的真核表达载体pcDNA3.1GFP,经酶切及测序鉴定后,用脂质体介导法转染小鼠精子,在荧光显微镜下观察并应用PCR技术检测HBV preS1基因的存在与表达情况。结果 成功构建了pcDNA3.1preS1GFP真核表达载体,将其转染小鼠精子后，在荧光显微镜下观察到转染精子头部有绿色荧光，PCR扩增得到位于HBV preS1基因区域内长约589 bp的片段。结论 重组质粒pcDNA3.1preS1GFP的成功建立及其在小鼠精子内的表达，为建立新的HBV DNA垂直传递的小鼠模型奠定了实验基础。 【关键词】 绿色荧光蛋白质类;质粒;肝炎病毒,乙型;精子;转染;小鼠 [ABSTRACT] Objective To construct pcDNA3.1preS1GFP recombinant eukaryotic expression vector harboring green fluorescent protein (GFP) and hepatitis B virus (HBV) preS1 gene and observe the expression of sperm in mice. Methods The HBV preS1 gene fragment was amplified from the HBV genome plasmid by PCR and inserted into eukaryotic expression vector pcDNA3.1GFP. After the identification by digestion and sequencing, the recombinant eukaryotic expression vector was transfected into sperm of mouse by Lipofectamine TM2000. Expression of GFP was examined directly with fluorescence microscope. The existence and expression of HBV preS1 gene was detected by PCR. Results The eukaryotic expression vector was created successfully and transfected to sperm of mouse with pcDNA3.1preS1GFP recombinant plasmid, green fluorescence was observed with fluorescence microscope, which presented on the head of sperm, and a specific band at 589 bp tha in the area of HBV preS1 gene was obtained by PCR. Conclusion The successful construction of the pcDNA3.1preS1GFP recombinant plasmid and its expression in sperm of mouse establishes an experiment foundation for creating a new mouse model of verticaltransmission HBV DNA. 　　[KEY WORDS] green fluorescent proteins; plasmids; hepatitis B virus; sperm; transfection; mice 　　乙型肝炎是由乙型肝炎病毒(HBV)感染引起的一种在全球范围流行的传染病,严重危害人类的健康。HBV是影响人类生存最重要的病毒之一, 全世界至少有3.5亿人感染HBV,在我国，其在人群中的感染率已经达到10%。HBV是一种部分双链的环状病毒, 具有随机整合入宿主基因组中的能力[1]。垂直传播是HBV感染的重要传播途径之一,动物实验业已证实生殖细胞内HBV DNA可通过受精过程稳定地传给子代[2]。由于HBV感染具有明