【摘要】 目的利用分子克隆技术构建含人野生型(WT)及致病突变A30P(G88C)、A53T(G157A)α-突触核蛋白基因(α-synuclein gene,SNCA)的重组真核表达载体pLentiVENUS-YFP-SNCA,通过慢病毒转染的方法获得过表达人野生型及致病突变A30P、A53Tα-synuclein单克隆SH-SY5Y细胞株。方法提取红白血病细胞K562细胞总RNA,以RT-PCR法扩增SNCA,SNCA与克隆载体PMD-19T的体外连接(T-A克隆)后进行基因测序,将测序正确者以限制性内切酶酶切后与真核表达载体pLentiVENUS连接,建立野生型重组真核表达载体。取正常SNCA与克隆载体连接体,利用单核苷酸差异引物定点突变法构建SNCA的两个突变型A30P、A53T,经基因测序、酶切与真核表达载体pLentiVENUS连接,建立突变型的重组真核表达载体。以磷酸钙沉淀法转染293T细胞制备的慢病毒转染SH-SY5Y细胞,利用流式细胞仪BD AriaⅢ进行96孔板单细胞分选以获得稳定过表达人野生型及致病突变型A30P、A53Tα-突触核蛋白的单克隆细胞株,并通过倒置荧光显微镜、蛋白免疫印迹、逆转录-聚合酶链反应(reverse transcriotion-polymerase chain reaction,Rt-PCR)、鉴定各单克隆SHSY5Y细胞株是否过表达。结果 Rt-PCR及电泳结果显示所获得目的基因,基因测序结果正确;重组真核表达载体pLentiVENUS-SNCA经限制性内切酶酶切和基因测序证明构建成功。倒置荧光显微镜显示除对照组(未转染组)外,空载体转染组及载体与SNCA重组后转染组均有荧光蛋白表达;但蛋白免疫印迹结果显示载体与正常或突变的SNCA重组后转染组的蛋白含量高于空载体组。RT-PCR结果显示载体与正常或突变的SNCA重组后转染组的细胞RNA表达量高于空载体组。结论利用分子克隆技术和慢病毒转染技术成功建立过表达α-突触核蛋白的WT及A53T、A30P突变型SH-SY5Y单克隆细胞株。更多还原

【Abstract】 Objective To establish recombinant eukaryotic expression vector pLentiVENUS-YFP-SNCA including Wild-type(WT)and A30P(G88C),A53T(G157A)α-synuclein gene(SNCA)and monoclonal SH-SY5 Ycell lines overexpressing WT,mutation A30 Pand A53Tα-synuclein by molecular cloning techniques and lentivirus infection method.Methods SH-SY5 Ycells overexpressing WTα-synuclein was generated,RNA extracted from k562 cells was reversely transcribed with reverse transcriptase into cDNA,and PCR was used to amplify human SNCA.The gene was sequenced after T-A clone(in vitro ligation of SNCA and cloning vector PMD-19T)and then the right one was digested by restriction endonuclease.The right sequence was ligated into the eukaryotic expression vector pLentiVENUS-YFP. WT recombinant eukaryotic expression vector wasestablished.The familial PD-linked A53 Tand A30P mutation were generated by site-directed gene mutagenesis using primer difference in mononucleotide on the base of WT SNCA.We confirmed the orientation and sequence of each construction by restriction analysis and sequencing.We obtained the lentiviral vector containing the recombinant eukaryotic expression vector of pLentiVENUS-YFP-SNCAby calcium phosphate precipitate method in 293 Tcells.Then these lentiviral vectors infected SH-SY5 Ycells,and then positive cells were sorted by BD AriaⅢ.Finally,the expression levels ofα-synuclein were assessed by inverted fluorescence microscope,protein immunoblot,and RT-PCR.Results The results of PCR and gene sequence detection shown that we obtained objective gene.The recombinant eukaryotic expression vector pLentiVENUS-SNCA were built,and checked by restriction enzyme digestion and sequencing.Inverted fluorescence microscope showed that cells expressed fluorescence protein except nontransfected cells,which indicated plasmids expressed.The result of protein immunoblot and RT-PCR proved that the protein content and RNA expression level were higher in the cells transfected with SNCA than the empty vector group.Conclusions We successfully establish the monoclonal SHSY5 Ycell lines overexpressing WT,mutation A30 Pand A53Tα-synuclein by molecular cloning techniques and lentiviral vector infection.更多还原