汉坦病毒核衣壳蛋白基因与EGFP基因在BHK21细胞中的融合表达
发布时间：2019-02-28 12:46
【摘要】：目的 构建汉坦病毒(hantavirus,HV)莒南扩增株JUN05株核衣壳蛋白(nucleocapsid protein,NP)的荧光蛋白表达载体;将HV NP基因与EGFP基因在BHK21细胞中融合表达,荧光显微镜下观察融合蛋白的表达强度,以及融合蛋白在细胞中定位和分布,免疫组化观察融合蛋白的免疫反应性,从而为新型HV诊断试剂研制与开发,以及NP结构与功能的研究奠定基础。 方法 根据HV莒南扩增株JUN05株NP基因的序列和表达载体pEGFP-N1的多克隆位点序列,设计引物,通过PCR的方法扩增NP全基因。PCR产物回收后以核酸内切酶BglⅡ、PstⅠ双酶切,克隆至同样双酶切的pEGFP-N1载体上,并保持NP基因与EGFP基因的读码框一致,CaCl_2法转化并筛选阳性克隆。通过PCR方法和双酶切方法鉴定,并通过DNA测序技术最终鉴定表达载体的构建情况。将重组质粒以脂质体法转染BHK21细胞,转染24h后固定细胞,荧光显微镜检和免疫组化技术观察融合蛋白的表达并拍照记录。 结果 1.构建了含有HV NP基因的荧光蛋白表达载体,酶切鉴定、PCR鉴定、及测序结果表明载体构建成功。 2.测序结果表明,NP基因成功连入EGFP基因上游,二者读码框一致无移位,与NP基因在克隆载体中的序列比对,无PCR扩增导致的碱基突变,并且NP基因末端的终止密码子TAA被去除。并添加了kozak序列,为高效表达提供了基因水平的改造。 3.荧光显微镜下观察,NP-EGFP融合蛋白在BHK21细胞中得到高效表达,细胞浆内充满了致密的绿色荧光,荧光呈核周分布,达到了以绿色荧光蛋白作
[Abstract]:Objective to construct the fluorescent protein expression vector of nucleocapsid protein (nucleocapsid protein,NP) of Hantaan virus (hantavirus,HV) JUN05 strain. The fusion protein of HV NP gene and EGFP gene were expressed in BHK21 cells. The expression intensity of fusion protein, localization and distribution of fusion protein were observed under fluorescence microscope, and the immunoreactivity of fusion protein was observed by immunohistochemistry. It lays a foundation for the research and development of a new HV diagnostic reagent and the study of the structure and function of NP. Methods according to the sequence of NP gene of JUN05 strain amplified by HV and the polyclonal site sequence of expression vector pEGFP-N1, primers were designed and the whole NP gene was amplified by PCR method. The products were digested by Bgl 鈪，
本文编号：2431808
[Abstract]:Objective to construct the fluorescent protein expression vector of nucleocapsid protein (nucleocapsid protein,NP) of Hantaan virus (hantavirus,HV) JUN05 strain. The fusion protein of HV NP gene and EGFP gene were expressed in BHK21 cells. The expression intensity of fusion protein, localization and distribution of fusion protein were observed under fluorescence microscope, and the immunoreactivity of fusion protein was observed by immunohistochemistry. It lays a foundation for the research and development of a new HV diagnostic reagent and the study of the structure and function of NP. Methods according to the sequence of NP gene of JUN05 strain amplified by HV and the polyclonal site sequence of expression vector pEGFP-N1, primers were designed and the whole NP gene was amplified by PCR method. The products were digested by Bgl 鈪，
本文编号：2431808
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