25714.嗜麥芽寡養(yǎng)單胞菌d2株堿性磷酸酶基因的克隆與表達

1、青島大學碩士學位論文嗜麥芽寡養(yǎng)單胞菌D2株堿性磷酸酶基因的克隆與表達姓名：楊麗申請學位級別：碩士專業(yè)：臨床檢驗診斷學指導教師：閆志勇20100603CloneandprokaryoticexpressionofStenotrophomonasmaltophiliaD2alkalinephosphataseAbstract0bjectiveWeobtainedaproteinhyperproducingStenotrophomonasma

2、ltophiliastrainfromthedigestivetractofPerinereisaibuhitensisGrubeAftertheproteinpurified，wesequenceditsaminoacidinOrdertogetthecodingsequencebybioinformaticsandMolecularBiologytechnologyWealsoexpressedthecodingsequencein

3、BL21toknowmoreaboutthesecretionandexpressionmechanismoftheD2MethodsThepurifiedD2alkalinephosphatasewasdigestedbyenzymes，andthensequencedbyESI—MS／MSWeblastedthosesixaminoacidfragments，andfoundthatthosefragmentshadhighiden

4、titiesofthestenotrophomonasmaltophiliaK279aandR55l3ThephosphatasegenewasamplifiedbythemethodofPCRwiththeprimerdesignedaccordingtotheK279aandl巧513whohadhigherIdentitiesThenweconjunctedthePCRproducttoTvectorandsequencedWea

5、lsoexpressedthegenewhichverificationbysequencinginEscherichiacoliBL21(DE3)byyieldinghybridplasmidpET32a，inducedwith口TG，andidentifytheres／dtbyWesternBlotWealsousedthe48hzymogenicsupematanttodetectthealkalinephosphataseact

6、ivity。ResultsWegota1438bpgenesequenceofthestenotrophomonasmaltophiliaD2，thephosphatasegenecontainedasingleORFspanning1233bp，anditsGeneBanknumberwasGQ413951WetranslatedtheORFint0aminoacidsequence，andcompareditwiththesteno

7、trophomonasmaltophiliaK279aandR5513alkalinephosphataseTheiridentitieswere685％and736％WealsocomparedthephosphataseaminoacidsequencewiththeaminoacidfragmentswhichsequencedbyESI—MS／MS，theidentitieswere100％，100％，632％，778％，889

8、％and421％TheORFencodeda66KDproteinWesternBlotindicatedthattherecombinantproteinistheD2alkalineproteaseWealsofoundthesequenceofthefirst20aminoacidinthephosphataseaminoacidactedassignalpeptide，whichmayplayanimportantroleint

9、heproteinIsecretionThe48hzymogenicsupematanthadalkalinephosphataseactivity，theactivitieswas1879ConclusionwegainedthegeneofD2alkalinephosphatase，andsuccessf；ulexpresseditinEscherichiacolineproteinhadphosphataseactivity，it

10、mightbeanewsourceofphosphataseofmarinemicroorganisms，andanewexpressionsystemPostgraduate：YANGLi(ClinicalLaboratoryDiagnostics)AdvisedbyProfYANZhiYongKeywords：StenotrophomonasMaltophilia；AlkalinePhosphatase；Clone；expressi