人DC-SIGN真核表达载体的构建及其在A549细胞中的表达

Construction of Eukaryotic Expression Vector of DC-SIGN and Its Expression in A549 Cells

目的:构建人DC-SIGN基因真核表达质粒,观察其在人肺腺癌细胞A549中的表达。为进一步研究DC-SIGN的作用奠定实验基础。方法:用PCR的方法扩增编码DC-SIGN的基因序列,将其克隆到真核表达载体pCDNA中,酶切及测序鉴定重组质粒。将构建的重组质粒转染到A549细胞中,用Western Blotting和免疫荧光等方法检测DC-SIGN基因的表达。结果:从人cDNA文库中得到1 215bp的DC-SIGN序列后,重组到pCDNA载体中,经酶切及测序鉴定,成功构建pCDNA-DC-SIGN重组质粒。重组质粒转染A549细胞,经Western blotting检测,发现在约55kDa处有特异条带,与理论大小相符。应用免疫荧光技术检测DC-SIGN可在A549细胞内的表达。荧光显示Myf5蛋白定位在细胞浆中。建立表达DC-SIGN的细胞株。结论:成功构建了人DC-SIGN的真核表达载体,并建立了人DC-SIGN的真核表达细胞株。

Objective：To construct a vector of pCDNA-DC-SIGN for studying of DC-SIGN protein.Method：The full length gene of DC-SIGN was amplified by PCR.Then it was cloned into pCDNA eukaryotic expressing vector.The recombinant DC-SIGN expressing construct was confirmed by using EcoRⅠand BamHⅠ double digestion and by sequencing.The pCDNA-DC-SIGN was transfected into A549 cells.The expression of DC-SIGN was examined using immunofluorescence technical and western blotting assay.Result：DC-SIGN which was about 1 215bp and obtained by PCR was recombinant into pCDNA vector.pCDNA-DC-SIGN eukaryotic expression vector was successfully constructed.Through Western blotting we find there is a distinctive band about 55kD,which identical to theoretical size.Specific expressions of DC-SIGN were detected with immunofluorescence technical in A549 cells and DC-SIGN protein was found locating inhyalomitome.Conclusion：DC-SIGN was successfully constructed into pCDNA eukaryotic expressing vector,and established DC-SIGN-expressed cell line.