TRAIL的基因克隆及其表達條件優(yōu)化研究.pdf

1、浙江大學(xué)生命科學(xué)學(xué)院碩士學(xué)位論文TRAIL的基因克隆及其表達條件優(yōu)化研究姓名：周銀燕申請學(xué)位級別：碩士專業(yè)：微生物學(xué)指導(dǎo)教師：吳雪昌20080601浙江大學(xué)碩士學(xué)位論文AbstractTumornecrosisfactorrelatedapoptosis—inducingligand(TRAIL)selectivelyinducesapoptosisinnumerousttullorcelllineswhilesparingnormal

2、cellsOnceitofficiallyputintouse，largequantitiesofbiologicallyactiveTRAILareneededAshasbeenreported，TRAILmainlyexpressedintheformofinclusionbodyWhereas，thelowefficiencyandcomplicatedprocessofrenaturation，whichisinevitable

3、fortheinclusionbodytogetfunctional，obstructsitsmassproductionTherefore，reducingtheinclusionbodyandincreacingthesolubleproteinwillpromotetheindustrialdevelopmentofTRAILInthisstudy，theeDNAsequenceofTRAIL(encodingresidues11

4、4281)wasamplifiedbyPCRThen，thegeneswereclonedintoplasmidpET28aandexpressedinEcoliBL21(DE3)WesternblotanalysisshowedspecificreactionoftheexpressedrecombinantproteinTheresponsesurfacemethodology(RSM)Wasadoptedtooptimizethe

5、cultureconditionsofTRAILTheproductionofsolubleTRAILwasimprovedfrom14684mg／Lto45485mg／LafteroptimizationFurthermore，hilgh—densityfermentationwasperformedProductionofsolubleTRAILconcentrationreachedto366egLunderthefermenta

6、tionconditionsBysonicationandeentrifugation，thesupernatantWascollectedandloadedontochromatographyforpurificationTRAILwerepurifiedbyNiaffinitychromatographyandion—exchangechromatographycombinational1049／LofpurifiedTRAILwa

7、sobtainedafterpurificationFinally，thepurifiedsolubleTRAILWaSidentified，andtheresultsshowedthattherecombinantproteinwasintheconditionofhighpurity，higllactivityandthemolecularweightmatchedwell、析tllthetheoreticalvalueKeywor