56067.基因工程白蛋白在畢赤酵母中的高效表達(dá)

1、華中農(nóng)業(yè)大學(xué)碩士學(xué)位論文基因工程白蛋白在畢赤酵母中的高效表達(dá)姓名：宋敏申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：生物化學(xué)與分子生物學(xué)指導(dǎo)教師：鄧子新鄭應(yīng)華2002.5.1AbstractTostudytheexpressionofrecombinanthumanseiumalbumininpichiapastorisandtOobtainhigh。levelexpressionofrHSAwithgoodbiologicalactivityNumerou

2、sfactorsthatinfluenceheterologousproteinproductioninacertainhostsystemmustbeobservedtogainsuccessfulgeneexpressionandevenwithhighyields，inpichiapastoristheyincludes：propeniesofheterologousgene，siteandmethodofintergration

3、oftheexpressioncassette，methanolutilizationphenotypeofthehostgenedosage，stabilityoftheproduct0urresultshowedthatthesecretingofrHSAinpichiapastoriswasashighasO599／LRapidoptimizationofmicrobiologicalmediabyPlachettBurman，a

4、fulleranalysis，togetherwithsomecorrespondingstrategiesand／orsuggestionwasdonein5LNBSBatch／continuousbioreactorcultureOnthebasisoftheexperimentstheotherfactorssuchasoptimalmethanolfeedingstrategy，initialpH，optimalglycerol

5、feedingstrategywerealsomentionedInadditiontheinfluencesoftheinitialpHonproteaseactivitywereinvestigatedUsingtheimprovedmediumtheconcentrationofrHSAinthefermentation1iquidreachedabove3509／LThesupernatantwasheatedat60℃for8

6、hoursandcentrifugalizedtogetridofthesedimentTheheat—treatedsolutionwastreatedwithanultrafiltrationmembranehavingamolecularweilghtexclusivelimitof30000t11enaddingaminosulphatetoafinalconcentrationof80％toprecipitaterHSAThe

7、precipitatedrhsawasseparatedfromthesupematantfluidbycentrifufation，redissolvedwithdidistilledwaterThealbuminsolutionobtainedintheaboveprocessingwasappliedtoacolmnnpackedwithPhenylsepharoseRTM，thenthealbuminelutedfromthec

8、olumnWasappliedtoacolumnpackedwithDEAESephadexRTMThelinearelutionwascarriedoutandrHSAcouldbeeluteddownafterhaIfofthelinearTherHSAsolutionobtainedafterthepurificationprocedurebyion—exchangechromatographywasanalyzedbymeans

9、ofHPLCAsshownintheresultthepurifiedrHSApreparationwasfoundasasinglepeakofrHSAmonomerThepresenttechnologicalprocessiSsimpleandconvenientandrHSAthusobtainedmaybehopefulofpassingthroughthestandardofC∽KeyWords：rHSA，Pichiapas