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人HMGB1基因原核表达载体的构建与表达.docx

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人HMGB1基因原核表达载体的构建与表达 Title:ConstructionandExpressionofaProkaryoticExpressionVectorfortheHumanHMGB1Gene Abstract: HighMobilityGroupBox1(HMGB1)proteinplaysacrucialroleinvariouscellularprocesses,includingDNAreplication,transcription,andrepair.Therefore,understandingthemechanismsofHMGB1geneexpressionisessentialforelucidatingitsbiologicalfunctionsandpotentialtherapeuticapplications.Inthisstudy,weaimedtoconstructaprokaryoticexpressionvectorforthehumanHMGB1geneandevaluateitsexpressioninEscherichiacoli. Introduction: TheHMGB1geneencodesahighlyconservednuclearproteinthatactsasaDNAchaperone,bendingandunwindingDNAstructurestofacilitatevariouscellularprocesses.PreviousstudieshaveidentifiedtheimportanceofHMGB1incancerprogression,inflammation,immunity,andtissueregeneration.ToinvestigatethefunctionofHMGB1,itiscrucialtoestablishanefficientexpressionsystemforproteinproduction. Methods: 1.IsolationandamplificationoftheHMGB1gene:TheHMGB1genewasisolatedfromhumangenomicDNAbyPCRusingspecificprimers.TheamplifiedDNAfragmentwasthenpurifiedandverifiedbysequencing. 2.Constructionoftheprokaryoticexpressionvector:ThepurifiedHMGB1genewasligatedintoaprokaryoticexpressionvector,suchaspET-28a(+),usingspecificrestrictionenzymes.TheresultingconstructwastransformedintocompetentEscherichiacolicellsusingheatshockandplatedontoselectivemedia. 3.Verificationoftheexpressionvector:ThepresenceoftheHMGB1geneintheconstructedvectorwasconfirmedbyrestrictionenzymedigestionandsequencing.Thecorrectvectorwasselectedforfurtheranalysis. 4.Proteinexpressionandpurification:TheselectedHMGB1expressionvectorwastransformedintoexpression-competentEscherichiacolicellsandinducedwithIPTG(isopropylβ-D-1-thiogalactopyranoside)toinitiateproteinexpression.TheexpressedHMGB1proteinwasthenpurifiedusingaffinitychromatographyorotherpurificationtechniques. 5.CharacterizationoftheexpressedHMGB1protein:ThepurifiedHMGB1proteinwasanalyzedbySDS-PAGE(sodiumdodecylsulfate-polyacrylamidegelelectrophoresis)todetermineitsmolecularweightandconfirmit