Build A Stable Expression Of Inflammatory Factors Of Tnf¦Á, Il-1¦Â In Cell Lines

Alzheimer's disease is a kind of age-related neurodegenerative diseases,leading to progressive loss of cognitive function.In the pathological process of AD,microglial cells and astrocytes are activated to produce a product of a series of immune responses.Tumor necrosis factor-α(TNF-α),IL-1β(IL-1β) have high expression.Pre-group research has confirmed:AD model rat brain hippocampus and prefrontal cortex areas,levels of inflammatory cytokines TNF-αand IL-1βwere significantly increased.After drug treatment in hippocampus and frontal cortex of IL-1βand TNF-αwere reduced significantly.According to literature at home and abroad,with the exception of AD,in other encephalopathy,the brain has different degrees i of inflammatory response,and inflammatory cytokines TNF-αand IL-1βare widespread,and enjoy a large number of expression.Therefore,inflammatory cytokines TNF-αand IL-1βas our object of study,in the brain,the universal significance.The contents of my experiment,in vitro,is to establish a highly efficient expression of inflammatory cytokines TNF-αand IL-1βin cell lines.On this base,the mechanism can be further researched,which provide a guideline to molecular pharmacology in drugs.Objective:Establishment of high level expression of inflammatory cytokines(TNFα,IL-1β) of the cell lines.Methods:1.To clone the target genes of TNF-αand IL-1βfrom vectors pCMVSport-TNF-αand the pCMVSport-IL-1β.Using of enzymes to carry out high-fidelity polymerase chain reaction(PCR),a large number of access to the target gene(that is,inflammatory cytokines TNF-αand IL-1βgene sequences)2.Eukaryotic expression vector pFLAG-CMV,pcDNA3.1,and amplified the target gene TNF-α,IL-1β,respectively,for dual-enzyme reaction,and 16℃,overnight reaction to connect to the target gene TNF -α,IL-1β,respectively,even into the eukaryotic expression vector pFLAG-CMV,pcDNA3.1;3.Three pairs of single-enzyme digestion of recombinant plasmid(compared with the empty vector),the recombinant plasmid and the PCR reaction of the base gene sequencing to identify whether the recombinant plasmid was constructed successfully, that is,the recombinant plasmid pFLAG-CMV-TNF-α,pFLAG-CMV-IL-1β, pcDNA3.1-TNF-α,pcDNA3.1-IL- 1β.4.Select the HEK293 cells,stable transfection using the method to eukaryotic expression vector pFLAG-CMV-TNF-αand pFLAG-CMV-IL-1β,the use of liposome-mediated transfection method of eukaryotic cells HEK293.48 hours after transfection,selection for resistance to G418,G418,after adding the first two days,significant changes in cell morphology;adding G418 after six days,a large number of dead cells began to appear; adding G418 after 10 days,there monoclonal cells,and cells in the digestion to 48-well plates to ensure that one cell per hole,G418 concentration to maintain the concentration, that is,half the concentration of screening check;adding G418 after 21 days,as long as monoclonal cell colony and show island;to continue to foster a,1-2 weeks,cells covered the entire culture hole digest cells,passaged or frozen cleavage detection.5.Use the method of western blot to test the levels of TNF-αand IL-1βexpression.Firstly, to cleavage cells,and use the standar protein production to draw a standard curve of protein,then calculate the volume of the sample for the detection of the target protein. Results:1.To clone vector pCMVSport-TNF-αand the pCMVSport-IL-1βfor the amplification of the template successfully amplified the target gene.Agarose gel electrophoresis results showed that target gene TNF-αand IL-1βin the expected position,that is,the size of the location of TNF-αin the 700bp,IL-1βin the size of the location of 800bp.Known TNF-αgene size of 701 bp,IL-1βsize in bp.2.Recombinant plasmid was successfully constructed.Recombinant plasmid pFLAG-CMV-TNF-α,pFLAG-CMV-IL-1β,pcDNA3.1-TNF-α,pcDNA3.1-IL-1βby the restriction endonuclease Hindâ…¢digestion alone,PCR reactions,as well as the reorganization of the quality tablets on gene sequencing of target genes.The target gene nucleotide sequence in GeneBank results hTNF-αand hIL-1βfor sequence alignment,use the DNAStar software than on the analysis,further confirmed that even the accuracy of the target gene into the eukaryotic expression vector.Known GeneBank sequence of hTNF-αnumber NM₀00594,hIL-1βsequence number NM₀00576.3.The stability of eukaryotic cell expression of inflammatory cytokines TNF-αand IL-1βsuccessfully established cell lines.The use of recombinant plasmid pFLAG-CMV-TNF-αand pFLAG-CMV-IL-1β,the use of liposome-mediated method of HEK293 cells stable transfection.Among them,the chromosome of the recombinant plasmid into HEK293 cells with G418 in the complete culture to survive,while the occurrence of transient transfection of cells,the recombinant plasmid that is free in the cytoplasm of HEK293 cells,resistance screening in the first 6 days while a large number of deaths.Lines result,the three stable transfection of the pFLAG-CMV into HEK293 cells,respectively,label pf1,pf2,pf3;6 strain stable transfection into the pFLAG-CMV-TNF-αin HEK293 cells,respectively, numbered T1,T2,T3,T4,T5,T6;5 strain stable transfection into the pFLAG-CMV-IL-1βin HEK293 cells,respectively,numbered I1,I2,I3,I4,I5.4.Using western blot to test the expression of inflammatory cytokines.First of all,the protein,the formula for the standard curve y = 2203.9x-790.85,of which R²=0.985. Western blot test results showed that cell line T1,T3,T4 can be higher expression of inflammatory cytokines TNF-α;cell line I2,I3,I5 can be higher expression of inflammatory cytokines IL-1β.Conclusion:1.Using high-fidelity PCR enzymes for amplification,can be accurate from the cloning vector pCMVSport-TNF-αand the pCMVSport-IL-1βon the amplified inflammatory factors TNF-αand IL-1βgene sequence and the sequence in GeneBank unanimously.2.Recombinant plasmid was successfully constructed,as follows:First,even the accuracy of the target gene into the eukaryotic expression vector;â…¡gene in the eukaryotic expression vector has been translated into the gene that is not even the "reading frame" sequence correct.3.Stable expression of inflammatory cytokines TNF-αand IL-1βlines of the successful cell lines.Recombinant plasmid DNA into HEK293 cell chromosome,the target gene can be effectively expressed in HEK293 cells and cells to the next generation will be genetic.