自噬作用能够增强猪瘟病毒在体外的复制

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1、TherelationshipbetweenviralandautophagyrestrictedSindbisvirusTobaccomosaicvirusinhibitHSV-1HIV-1utilizecoxsackievirusdenguevirusinfluenzaAvirushepatitisCvirusCSFVAutophagyMethodfordetectingautophagy自噬性结构的电镜下形态学观察基于自噬体标记蛋白LC3的检测方法基于自噬降解的检测自噬的实验性调控自噬检测的金标准透射电子显微镜是观

2、察自噬现象的最直接、最经典的方法，电镜检测自噬主要是基于辨认自噬体结构。免疫金电镜技术来对电镜结果进行定量分析．通过图像分析软件自动测量所有自噬囊泡的面积，在结合其他检测方法的基础上，如果一个细胞胞浆中被自噬性囊泡所占据的总面积增加，则可说明自噬机制的上调。基于自噬体标记蛋白LC3的检测方法微管相关蛋白1轻链3(LC3)是自噬体膜上标记蛋白．细胞内存在两种形式的LC3蛋白：LC3Ⅰ和LC3-Ⅱ．LC3蛋白在合成后其C端即被Atg4蛋白酶切割变成LC3-Ⅰ(细胞浆内)．当自噬体形成后，LC3-Ⅰ和PE偶联形成LC3-Ⅱ(自噬

3、体膜)．LC3-Ⅱ始终稳定地保留在自噬体膜上直到与溶酶体融合，LC3-Ⅱ的水平在某种程度上反映了自噬体的数量.celllinesPK-15themodelcelllineforstudyingCSFVinfection.3D4/2celllinerepresentativeofthemacrophagethatisthetargetforCSFVinfection.ConsideringthenoncytopathogenicnatureofCSFVidenticalmultiplicitiesofinfection(MO

4、Is)andinfectiontimepointswereusedforbothPK-15and3D4/2cells.CSFVinfectionincreasestheformationofautophagosome-likevesicles(A)PK-15and3D4/2cellsweremock-infectedorinfectedwithCSFVatMOIof1for48h.Whitearrowsindicatethestruct–uresofautophagosomes.LC3proteinimmunogoldla

5、belinglocalizedtotheinfection-associatedmembranesisindicatedbyblackarrowsintheinfectedcells.(B)Quantificationoftheautophagosome-likevesiclespercellimage.expressionofautophagymarkerproteinsinCSFV-infectedcellsPK-15cellsweremock-infectedorinfe–ctedwithCSFVorUV-inac

6、tivatedCSFVfor6,12,24,36,and48h.theexpressionofLC3,ATG12–ATG5,ATG5,BEDN1,E2,ACTB(loadingcontrol)wereanalyzedbyimmuno-blottingwithspecificantibodies.(B)3D4/2cellswereinfectedandanalyzedasin(A).CSFVinfectioninducestheredistributionoftheautophagymarkerLC3(A)PK-15cell

7、sweremock-infectedorinfectedwithCSFVorUV-inactivatedCSFVortreatedwithrapamycinfor48h.Thecellswereprocessedforindirectimmunofluore-scence.ThecellnucleuswerecounterstainedwithDAPI.Thenucleusstainingisshowninblue,E2andNS5Astainingisshowningreen,LC3stainingisshowninre

8、d,andthesignalsofcolocalizationareshowninyellowinmergedimage(B)3D4/2cellswereinfectedandanalyzedasin(A).inductionofautophagywithrapamycinenhancesCSFVrep