重組人糖皮質激素受體原核表達載體的構建和表達.pdf

1、第二軍醫(yī)大學碩士學位論文重組人糖皮質激素受體原核表達載體的構建和表達姓名：王喜申請學位級別：碩士專業(yè)：中西醫(yī)結合臨床指導教師：凌昌全20050501重組人糖皮質激素受體原核表達載體的構建和表達摘要目的：構建hGRa重組原核表達質粒并進行誘導表達，為r一步研制hGRetELISA檢測試劑盒和進一步研究GR與葉醫(yī)學陰陽虛讓的關系服務。方法：以含有hGRaeDNA的重綁克隆質粒pRShGR|【t為模板，通過PER擴增得到hGRa的炳種基聞片段

2、，將這歷種基因片段分別克隆到pGEMT載體上進而亞克隆到pGEX4T3表選載體上。將這兩種重組表選質粒進行酶切、測序鑒定后，分別轉化大腸桿菌DHSct和BL21(DE3)，并在一定的條件下進行重組蛋白的誘導表達，誘導表達產物行SDS—PAGE和Westernblofung分析。結果酶切和測序結果證明hGRa的兩種基崮片段均成功地重組八pGEX一4T3載體。誘導表達產物的SDSPAGE和Westernblotting分析均提示有hGRaU

3、的蛋白特異表達。結論；車實驗成功構建了hGRa的歷種重組原枝表達質粒，并成功誘導，它們的原核表達，為卜一階段的研究奠定了基礎。關鍵司：陰陽虛讓；糖皮質激素受體；DNA重組技術：原核表達載體：蛋白表達ConstructionandexpressionoftheprokaryoticexpressionvectorofrecombinanthumanglucocoHticoidreceptorAbstrnetobjeetive：7locon

4、structhGRctrecombinantprokaryeticexpressionvectorandinducentoexpressThisvorkmightsorvethecreatingofhGR口ELISAdetectionkitandthefurtherclinicalstudyofcorrelationbetweenglueocorticnidreceptorandYhvYangdeficineysyndromeMetho

5、ds：。IwokindsofgenefragmentsofhGRawereamplifiedbyPCRfromtherecombinantcloningplgsmidpRShGRa，whichcarryingthehGRueDNAsequenceAfterbeingrespectivelyclonedintothepGEM—TvectorfirstthetWOkindsofgenefragmentswePesubelonedintoth

6、epGEX一4T3expressionvectoLThetwokindsofrecombinantexpressionvectorsweretransfumacdintotheEscheriekiacoliDH50findBL21fDE3)respectivelyafterbeinganalyzedbyrestrictionendonudeasedigestionandsequencingTheengineeringEcoliwerei

7、nducedtoexpressthefusionproteinsundersomeinductionconditionsandtheinducedproductsWereobserved、vithSDSPAGEandWesternblottingResults：Res)rictionendenucleasedigestionandsequencinganalysisshowedthatthetwokindsofgenefragments

8、ofh(JRawererecolnbinedintopGEXMT3vectorsuccessfullyTheSDSPAGEandWesternblo“inganalysisoftheinducedproductsshowedthatthetwokindsofhGRⅡtargetpru|teinswereexpressedspecificallyConelualon：Thetv、okindsofrecombinantexpresslonv