重組人糖皮質(zhì)激素受體原核表達(dá)載體的構(gòu)建和表達(dá).pdf

1、第二軍醫(yī)大學(xué)碩士學(xué)位論文重組人糖皮質(zhì)激素受體原核表達(dá)載體的構(gòu)建和表達(dá)姓名：王喜申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：中西醫(yī)結(jié)合臨床指導(dǎo)教師：凌昌全20050501重組人糖皮質(zhì)激素受體原核表達(dá)載體的構(gòu)建和表達(dá)摘要目的：構(gòu)建hGRa重組原核表達(dá)質(zhì)粒并進(jìn)行誘導(dǎo)表達(dá)，為r一步研制hGRetELISA檢測(cè)試劑盒和進(jìn)一步研究GR與葉醫(yī)學(xué)陰陽虛讓的關(guān)系服務(wù)。方法：以含有hGRaeDNA的重綁克隆質(zhì)粒pRShGR|【t為模板，通過PER擴(kuò)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(jìn)而亞克隆到pGEX4T3表選載體上。將這兩種重組表選質(zhì)粒進(jìn)行酶切、測(cè)序鑒定后，分別轉(zhuǎn)化大腸桿菌DHSct和BL21(DE3)，并在一定的條件下進(jìn)行重組蛋白的誘導(dǎo)表達(dá)，誘導(dǎo)表達(dá)產(chǎn)物行SDS—PAGE和Westernblofung分析。結(jié)果酶切和測(cè)序結(jié)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(dǎo)表達(dá)產(chǎn)物的SDSPAGE和Westernblotting分析均提示有hGRaU

3、的蛋白特異表達(dá)。結(jié)論；車實(shí)驗(yàn)成功構(gòu)建了hGRa的歷種重組原枝表達(dá)質(zhì)粒，并成功誘導(dǎo)，它們的原核表達(dá)，為卜一階段的研究奠定了基礎(chǔ)。關(guān)鍵司：陰陽虛讓；糖皮質(zhì)激素受體；DNA重組技術(shù)：原核表達(dá)載體：蛋白表達(dá)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