感觉神经元特异性受体rMrgD基因的克隆与表达

目的克隆rMrgD基因并于HEK293细胞中表达。方法利用聚合酶链反应技术,从总RNA中扩增出目的基因,克隆入真核表达载体pcDNA3.1中,构建真核表达rMrgD载体,瞬时转染人胚肾293细胞得到重组质粒表达细胞株。通过免疫荧光和Western印迹检测对表达产物进行鉴定。结果直接和间接免疫荧光实验皆表明rMrgD主要定位于细胞膜上,Western印迹检测证实rMrgD在HEK293细胞主要以单体和二聚体形式存在。结论含有rMrgD基因的重组质粒在人胚肾293细胞中获得表达,主要定位于细胞膜上。本研究为进一步探究rMrgD受体的功能奠定了基础。

Cloning and expression of sensory neuron-specific receptor rMrgD

Objective To clone the rat rMrgD gene and construct a new cell culture system to express rMrgD fusion protein.Methods By polymerase chain reaction （PCR）,the rMrgD gene was amplified from total RNA,cloned into pcDNA3.1 eukaryotic expression vector,and transiently transfected into Human Embryonic Kidney 293（HEK293）cells to produce the fusion protein positive cell lines.The expressed rMrgD could be identified with immunofluorescence and Western-blot.Results The rMrgD fusion protein had epitope which could be recognized by tag antibody c-myc and n-HA in HEK293.The rMrgD was mainly located in the cell membrane in the form of monomer and dimer.Conclusion The rMrgD is expressed in HEK293 cells and located in the cell membrane,facilitating further studies on the function of rMrgD.