波形蛋白过表达质粒pcDNA3.1b-Vim的构建及鉴定
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摘要
目的以波形蛋白(vimentin)基因为靶基因,构建波形蛋白过表达质粒pc DNA3.1b-Vim。方法在PC12H细胞中抽提RNA,进行逆转录聚合酶链式反应(RT-PCR),得到c DNA,设计引物将目的基因扩增并进行胶回收,将空载质粒pc DNA3.1b与目的基因片段进行双酶切(分别为EcoRI/Xba I)、连接,构建重组过表达质粒,转化到Top10感受态细胞,经氨苄青霉素筛选挑取阳性克隆,扩大培养,提取质粒,凝胶电泳鉴定并进行测序分析。结果凝胶电泳鉴定及DNA测序分析显示重组质粒pc DNA3.1b-Vim构建成功。结论成功构建波形蛋白过表达质粒pc DNA3.1b-Vim,为研究波形蛋白的生物学功能打下基础。
Objective Take the gene of vimentin as the target gene to construct the recombinant vimentin over-expressed plasmids. Methods mRNA was extracted from PC12 H cells and c DNA was obtained by reverse transcription polymerase chain reaction( RT-PCR).The special primers of vimentin were designed to amplify the target gene by PCR. No-load plasmid pc DNA3. 1 b and the target gene fragment were digested by enzymes( EcoRI/Xba I),and then to connect. The recombinant vimentin over-expressed plasmids were transformed into Top10 competent cells and screened by ampicillin. The plasmid was cultured,extracted,identified by gel electrophoresis and sequenced.Results The gel electrophoresis assay and DNA sequencing analysis showed that the recombinant vimentin over-expressed plasmids were constructed successfully.Conclusions The recombinant vimentin over-expressed plasmids were constructed successfully,which provides a foundation for further study on the biological function of vimentin.
Objective Take the gene of vimentin as the target gene to construct the recombinant vimentin over-expressed plasmids. Methods mRNA was extracted from PC12 H cells and c DNA was obtained by reverse transcription polymerase chain reaction( RT-PCR).The special primers of vimentin were designed to amplify the target gene by PCR. No-load plasmid pc DNA3. 1 b and the target gene fragment were digested by enzymes( EcoRI/Xba I),and then to connect. The recombinant vimentin over-expressed plasmids were transformed into Top10 competent cells and screened by ampicillin. The plasmid was cultured,extracted,identified by gel electrophoresis and sequenced.Results The gel electrophoresis assay and DNA sequencing analysis showed that the recombinant vimentin over-expressed plasmids were constructed successfully.Conclusions The recombinant vimentin over-expressed plasmids were constructed successfully,which provides a foundation for further study on the biological function of vimentin.
引文
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[14]Ma J,Hart GW.O-Glc NAc profiling:from proteins to proteomes[J].Clin Proteomics,2014,11(1):8.
[2]Ivaska J.Vimentin:central hub in EMT induction[J].Small GTPases,2011,2(1):51-53.
[3]Nieminen M,Henttinen T,Merinen M,et al.Vimentin function in lymphocyte adhesion and transcellular migration[J].Nat Cell Biol,2006,8(2):156-162.
[4]Dave JM,Bayless KJ.Vimentin as an integral regulator of cell adhesion and endothelial sprouting[J].Microcirculation,2014,21(4):333-344.
[5]Hui SP,Nag TC,Ghosh S.Characterization of proliferating neural progenitors after spinal cord injury in adult zebrafish[J].PLo SOne,2015,10(12):e0143595.
[6]Gorris R,Fischer J,Erwes K L,et al.Pluripotent stem cell-derived radial glia-like cells as stable intermediate for efficient generation of human oligodendrocytes[J].Glia,2015,63(12):2152-2167.
[7]Liu Z,Li Y,Cui Y,et al.Beneficial effects of gfap/vimentin reactive astrocytes for axonal remodeling and motor behavioral recovery in mice after stroke[J].Glia,2014,62(12):2022-2033.
[8]Chang IA,Kwon KB,Park YC,et al.Permissive role of Cdc2activity induced from astrocytes in neurite outgrowth[J].JNeurochem,2013,125(2):214-224.
[9]Vinci L,Ravarino A,Fanos V,et al.Immunohistochemical markers of neural progenitor cells in the early embryonic human cerebral cortex[J].Eur J Histochem,2016,60(1):2563.
[10]Chang IA,Oh MJ,Kim MH,et al.Vimentin phosphorylation by Cdc2 in Schwann cell controls axon growth via beta1-integrin activation[J].FASEB J,2012,26(6):2401-2413.
[11]Kaasik K,Kivimae S,Allen JJ,et al.Glucose sensor O-Glc NAcylation coordinates with phosphorylation to regulate circadian clock[J].Cell Metab,2013,17(2):291-302.
[12]Hart GW,Slawson C,Ramirez-Correa G,et al.Cross talk between O-Glc NAcylation and phosphorylation:roles in signaling,transcription,and chronic disease[J].Annu Rev Biochem,2011,80:825-858.
[13]Myers SA,Daou S,Affar El B,et al.Electron transfer dissociation(ETD):the mass spectrometric breakthrough essential for O-Glc NAc protein site assignments-a study of the O-Glc NAcylated protein host cell factor C1[J].Proteomics,2013,13(6):982-991.
[14]Ma J,Hart GW.O-Glc NAc profiling:from proteins to proteomes[J].Clin Proteomics,2014,11(1):8.