文章摘要
刘明慧,王春梅,陈京.食欲素2型受体点突变体表达载体的构建[J].济宁医学院学报,2017,40(6):389-392
食欲素2型受体点突变体表达载体的构建
Construction of OX2R mutant expression vector
投稿时间:2017-11-09  
DOI:10.3969/j.issn.1000-9760.2017.06.001
中文关键词: OX2R;点突变;表达载体;HEK293T细胞
英文关键词: OX2R;Point mutation;Expression vector;HEK293T cells
基金项目:山东省自然基金项目(ZR2013CQ031,ZR2015CL021)
作者单位
刘明慧 曲阜师范大学, 曲阜 273165 
王春梅 济宁医学院神经生物学研究所, 济宁 272067 
陈京 济宁医学院神经生物学研究所, 济宁 272067 
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中文摘要:
      目的 构建带有黄色荧光蛋白突变体Venus标签的食欲素2型受体(Orexin type 2 receptor,OX2R)点突变的真核表达载体,拟用于后续明确突变位点在OX2R介导的信号通路中的作用。方法 先根据OX2R的基因序列设计带有点突变及酶切位点(Hind Ⅲ和Bam HI)的特异性引物,然后利用聚合酶链式反应(PCR)技术,以Human OX2R为模板,构建OX2R的突变体。用Hind Ⅲ和Bam HI对产物及质粒pVenus-N1进行双酶切,然后回收,连接,转化,获得重组的突变体质粒。并对重组质粒进行酶切与测序鉴定。最后将突变质粒转染HEK293T细胞,利用超高分分辨率显微镜观察突变质粒的表达及定位。结果 PCR扩增到预期的目的基因片段,酶切与测序结果显示OX2R的突变载体构建成功。超高分分辨率显微镜观察到突变质粒与野生质粒均可在HEK293T细胞膜上表达。结论 成功构建了OX2R点突变的表达载体,为进一步研究OX2R的关键位点及针对OX2R新药的开发奠定了基础。
英文摘要:
      Objective To confirm the key sites of OX2R in the signaling pathway mediated by OX2R receptor,the point mutant vectors of OX2R with Venus were constructed.Methods Specific primers with restriction sites (Hind Ⅲ and Bam HI) and mutant point were designed according to the gene sequence of OX2R.Then,the fragments containing mutant points were amplified by Polymerase Chain Reaction (PCR) using the plasmid Human OX2R as template.The PCR products were digested by enzyme Hind Ⅲ and Bam HI,and cloned into plasmid pVenus-N1.The mutant vectors of OX2R were identified by restriction enzyme and DNA sequencing.Finally,the mutants were transfected into HEK293T cells,and the expression of them was confirmed by ultra high resolution microscope.Results We got the fragments containing mutant points by PCR,and sequencing results showed the vectors were correct.The mutant plasmids and control plasmid were expressed on the member of HEK293T observed by ultra high resolution microscope.Conclusion The mutant expression vectors of OX2R were successfully constructed.The mutant vectors can be used to study the effect of key point in OX2R on the signal pathways and benefit to the development of new drugs.
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