【摘要】 构建了携带绵羊朊蛋白基因(PRNP)的重组真核转染质粒,通过脂质体转染试剂将其转染至神经胶质瘤细胞(C6),以期为进一步研究绵羊朊蛋白的生理功能和从细胞水平研究朊蛋白疾病的发病机制奠定基础。采用PCR扩增目的基因序列,纯化后将其克隆到带有增强型绿色荧光蛋白(EGFP)报告基因的真核表达载体pEGFP-N1中,对重组质粒pEGFP-PRNP做酶切鉴定。而后采用阳离子脂质体转染法将重组质粒转染到C6细胞,荧光显微镜观察。经鉴定,绵羊PRNP基因已克隆到真核表达载体pEGFP-N1中,成功地构建了重组pEGFP-PRNP质粒,并可稳定地在C6细胞中表达。本研究为外源朊蛋白在细胞中表达提供了平台,为进一步在细胞水平研究朊病毒疾病奠定了基础。更多还原

【Abstract】 To construct a kind of recombinant plasmid with ovine prion protein gene(PRNP) and transfect the plasmid into C6 cell line in order to study the physiological function of normal prion protein and the pathogenesis in prion diseases.The gene sequence encoding ovine prion gene was amplified by PCR and cloned into eukaryotic expression vector pEGFP-N1 with EGFP reported gene encoding enhanced green florescence protein,and then identified by restriction enzyme.The recombinant plasmid pEGFP-PRNP was transfected into C6 cell line by using lipofectin method.We successfully construct the expression vector of recombinant plasmid pEGFP-N1 and it can be steadily expressed in form of fused protein in C6 cell line.It is suggested that the expression of recombinant plasmids pEGFP-PRNP in C6 cell line will provide a platform for researching expression of prion in cell and further contribute to study on prion diseases at cell level in future.更多还原