抗菌肽dermaseptin s4及其突变体基因在大肠杆菌中的融合表达

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1、兰州大学硕上学位论文结果发现得到了特异性目的蛋白，透析后经紫外分光光度仪分析，获得了0．2mg／ml的融合目的蛋白。结论：同时成功获得DS4及其突变体K4．S4目的基因片段；成功构建了表达重组体pGEX-4T-1-DS4)及突变型表达重组体pGEX．4T．1．杨．S4；成功实现了其在大肠杆菌中的融合表达及纯化，为进一步研究抗菌肽DS4基因的功能和开发新的药物奠定了基础。关键词：抗菌肽DermaseptinS4；重叠延f0PCR；基因克隆；融合表达Ⅱ兰州大学硕士学位论文FusionExpressionofTheAntimicrobialPeptideDer

2、maseptinS4GeneandItsMutantGeneinEscherichiaOilABSTRACTOBJECTIVE：ToobtainoptimumAntimicrobialpeptideDS4geneanditsmutant瞄一S4geneusinggenesplicingbyoverlapextensionPCR(SOEPCR)methodconstructrecombinantpGEX-4T．1一DS4andpGEX-4T-1·K=4一S4，expressionandpurifi-cationoffusionproteininE．coli

3、BL21．METHODS：BasedonaminoacidsequenceandthepreferencecodonofE．coliinCodonUsageDatabase，makefulluseoftheDNAStarandOligo6．0bioloigicsoftwaretodesignofDS4geneandK4一S4gene，whichwereamplifiedthroughusinggenesplicingbyoverlapextensionPCR(SOEPCR)method．AndtheywereclonedintovectorPUC-18，

4、AfterrestrictionanalysisandDNAsequencing，thetargetgenewerefurtherligatedwiththefusionexpressionvectorpGEX-4T-1，byusingrestrictionanalysisandDNAsequencing,thepositiverecombinantplasmidweretransformedtoE．coliBL21(DE3)plySforIPTGinducedexpression．Byoptimizingexpressionconditioninclu

5、dingdifferentinductiontimeandtemperatureandIPTGconcentration，theresultswereobservedby15％SDS—PAGEandtheyrevealedthattheGST—DS4andGST．K4．S4fusionproteinweresuccessfullyexpressed．HarvestingthepositiveE．coliBL21andthesolubilitybodywereobtained，thenpurifiedthroughGSTrapFFcolumnandobta

6、inedtheinterestingprotein0．2mg／m1．RESULTS：1．DS4geneanditsmutantK4-S4geneobtainedusinggenesplicingbyoverlapextensionPCR(SOEPCR)method，whichabout103bp(includingrestrictionendonu—cleasessiteandprotectivebase)．2．TherecombinantPUC．18．DS4andPUC-18·K4一S4wereconstructedsuccessfully,nI兰州大

7、学硕十学位论文andsequencinganalysisindicatedthatDS4geneandK4-S4genewreobtainedcorrectly．3．TherecombinantpGEX-4T-1-DS4andpGEX4T-1一K4-S4wenconstructedSUCC-essfully,sequencinganalysisindicatedthattheligationwerecorrectandcouldinducetheproteinexpression．4．BothpGEX一4T一1·DS4andpGEX一4T一1一K4-S4

8、recombinantwereinducedby0．1mMIPTGfor5hou