【word】 空肠弯曲菌penner

【】 空肠弯曲菌penner 空肠弯曲菌penner pennero:19cjaA???????<?????? ???L?L???1?)???0?????????X?????????/???y?T???? ?????????<???????f????pennero:19cjaA????,?????????z????????,?????????????4????,?F ???????a???????X?N???a???wke?????????????f????pennero:19cjaA????,????pGEMTcjaA?<??????, ??EcoRI??XhoI?F???1????????pGEX4T1cjaA???z????????ke???????????4E.coliBL21?G????????, SDSPAGE????????????,?3GlutathioneSepharose 4B ???4????????ke?????M??????pGEX4T1cjaA?????? ??,???????????F10g/l??rGSTCjaA????????ke?????M??????pGEMTcjaA?<????????pGEX4T1 cjaA????????,????????????rGSTCjaA,?F?X???????f???????????a???wke ???8???????f????;cjaA????;???z????;????????;???????4 ?)???:????:R37???8?1???p:A?????4??:1006351X(2011)02008104 ConstructionoftheprokaryoticexpressvectorofcjaAgeneofcampylobacterjejuniXINGCongcong, LIZhenzhong,BAIXinli,XUFei,JIANShuo,LIXin,ZHAOZichun,GAOAimin,XUEFeng.Departmentof Neurology,theSecondHospitalofHebeiMedicalUniversity,Shijazhuang050000,China. Correspondingauthor:LIZhenzhong,Emai:lJohndocter@vip.163.com AbstractObjectiveToconstructprokaryoticexpressionvectorcarryingcampylobacter.jejunipennero:19 cjaAgene,expressitinE.coliandpurifyprotein.MethodsThecjaAgenewereamplifiedbyPCR.Theclone vectorpGEMTandexpressionvectorpGEX4T1withinsertedcjaAgenewereconstructed.Thetargetproteinwas expressedinE.coliBL21.TheproteinwasidentifiedandanalyzedbySDSPAGE.Thetargetproteinwaspurifiedby GlutathioneSepharose 4B .ResultsConstructedtheexpressionvectorpGEX4T1cjaA,purifiedtherecombinant protein.ConclusionTherecombinantplamidpGEMTcjaAandpGEX4T1cjaAhavebeensuccessfully constructedandtheGSTCjaAproteinispurified,whichlaysthefoundationforthefurtherdevelopmentofC.jejuni geneticengineeringvaccine. KeywordsCampylobacterjejun;iCjaAgene;Prokaryoticexpression;Vectorconstruction;Purifyprotein. ????????:???????N?????????]??????(30471919);?????????f?X???? ?????R(2006AA02A208);(863???R2006AA02A2080) ???o????5??????,???.?J???;???(???J??????????(???L?Lkp ???1?)kp????kp?????/kp???y?T),?@??(???0??);???????????????w?? ?,???G???????<?)??(????kp????);?????;???J????(???X) ?B?????o???1?),@63 ?????f????(campylobacterjejun,iCj)?B???????; ?4?m??????????????????kp????,?f?4?Y?n?????????? ??????kp???????J????????ke?????f?????P???G???? ?????????????????/?;?????;???s???c?????????9 ??(acutemotoraxonalneuropathy,AMAN),???3???? ?t???)???k?????? [1] ke???x????????????kp?m?????? ?T?????????f???????P?????f?????????f?????????? ???????????????<??ke ???P???4???:???F???F??????????,????T???? ???????;?????????????4????????????kp????kecjaA ?????f?????;????,CjaA??C?5???3???????????? ??,???:?3???????x???4??,?fABC???W???3?????? ?????M?:?<??,?W???3???3?????f???????P?:???s?) ?g?F????,CjaA???c?????????;,?f?????4???????? ?????i?c?M?:?<??ke ?(???????????f????pennero:19???????F?t?? ?<??????cjaA????,???4?G?????????????F?????P?? ???????????????f???????P?????????e?;?????????f ????pennero:19???P??,???T?????f???????P?????? AMAN????????ke ?????????? ??kp???? ?????f?????y?(??????????ke ??D5kpBL???3??GXT?y?o???;???J???? ?z????????????keTqDNy,yDN 81?T?G?????????V??2011???(19???(2?P :00000 :Emai:lJohndoctervip.1.com pennero:19E.coli H21PE41 aApolmeraseprobestA polymerase,dNTP,T4DNA??????,???N?;???1?? (EcoRI??XhoI),DNAMarker,???:????????Marker, ?)??Takara????;pGEMTKit?)??Promega????, DNA???????N?G???4???V??,???????3???E???????? ?V???)???p?H????????;IPTG,?n????????R250?)?? Sigma????;GlutathioneSepharose 4B ?)??GE????ke ??kp???? 1.??????????DNA:?:?????f????pennero:19 ??????CM0739??????,?????510%CO2,5%O2???? ???;?????),37????48min,???3DNA???????V?? ????C.jejunipennero:19??????DNA,???Z?:?’?’?? ???<??????180ng/ulke 2.????cjaA????:???nGenebank(?4??NZ AASL01000001)????cjaA???.,?3???j?a?z?????h??,?h ?????.???a,Pfor:AGAGCTAGAATTTCCTCCACAAGA; Prev:ATTTAGAGCAAAGAATTTAATCTTGCkePCR?????? ???F94?????;5min,94???;30??,53????30??, 72???z1min,30?6????,72???z10min,4????ke1% ?H?e???????b??????PCR????,?3DNA???????N???4?? ?V?????N???4PCR????ke 3.????pGEMTcjaA?????3??:?:???4??cjaA?? pGEMT?c4:1???X?????7????1h,???4E.coliDH5 ?P???p????,?3?C?c?3???c?u??LB?????????c???? ?3??pGEM4T1cjaA?v?;?<??,?3EcoRI???1?????? ???3?????pinvitrogen?????<??????ke 4.????pGEX4T1cjaA?????3??:???v?;?<???3 ??pGEM4T1cjaA?F?t??,??cjaACDS?F???????., ??EcoRI??XhoI?F???1?????????h??,?h?????.???a, Pfor:cgagaattcATGAAAAAAATACTTCTAAG;Prev: tatctcgagAATTTTTCCACCTTCAATCke????cjaA?????? ???N???4,?3EcoRI??XhoI?????1cjaA??pGEX4T1, ???N???4cjaA??pGEX4T1???c4:1???X??16???? 3h,???4E.coliDH5?P???p????,?3?C?c?3???c?u?? LB?????????c?????3??pGEX4T1cjaA?v?;?<??,?3 EcoRI??XhoI?????1?????????3?????pinvitrogen???? ?<??????ke 5.?????3???????????????????E???:??:?:?v?; ?<???????3??pGEX4T1cjaA???4E.coliBL21,???? ?v?;?<?????C?c?3???c?u??LB??????37220??/?: ???2????,?c1:100??????????30150??/?:???? ??6=,?3??????L??ITG???????? ,?????N??????,?3BS????????,?????????m???? ?????????o??,?:??????,?j?o,????,?3%?:???? ???YSDSG?b??,?3?n????????R5???????? ???y?????y???G???????????????????Eke 6.???????????4:??48000??/?:????15min ?N??????????200ml????,?3PBS????????,?????? ???m??????30min,?N???????????G???j?o??,?3?J?4 ??GlutathioneSepharose 4B ???9????????,?3PH8.8, 20mMGSH?.?y???.?y????????,?:???4???????Y SDSPAGE?b??,?3?n????????R250???????????y ?????y???G?:???????????????4????ke ???? 1.cjaA????????????(??1) M:DNAladder;1~2:cjaA???????????C ??1cjaA????PCR???? ???????f??????????DNA?F?t?????YPCR????ke PCR???????Y1%?H?e???????b???:??ke?b???????d ?FPCR?????????F???????C,?;?E????1.4Kbp?rke 2.?????3??pGEMTcjaA???1???<??????(??2) M:DNAmarker;1~10:pGEMTcjaA?3EcoRI???1 ??2?????3??PGEMTcjaA?????1?H?e?????????? ?????3??pGEMTcjaA?3EcoRI???1?G???k?; ?E????1.4Kbp???????=??,???GcjaA?????M?????? ?kpGEMT???B?<??????ke ??pGEMT?B?3?h?????T?<??????????cjaA,?3 DNSTR?????<??????????jDS ???.ke 3?????3??GXTj???1???<??????(??3) 82?T?G?????????V??2011???(19???(2?P A000.40.1mmol/P 4hP 10 PAE20 AApennero:19caAC .pE41caA M:Marker;1~3:pGEX4T1cjaA?3EcoRI??Xhol?????1 ??3?????3??pGEX4T1cjaA?????1?H?e?????????? ?????3??pGEX4T1cjaA?3EcoRI??XhoI???? ?1???k?;?E????840bp???????=??,???GcjaACDS?? ?.?????M???????kpGEX4T1???B?<??????ke ??pGEX4T1?B?3?h?????T?<??????????cjaA, ?3DNASTAR?????????????<???????3??ke 4.?????3?????????????????????E?:??(??4) M:???:????????Marker;1:?????G????;2:?????G?j?o;3:?????G???? ??4????????rGSTCjaA?????????????????ESDSPAGE???? ?v?;?<???????3??pGEX4T1cjaA???4E.coli BL21,??30150rpm/min?3???????F0.1mmol/L?? IPTG????????4h,?:??????,?j?o,????,?B??SDS PAGE?n?????????????:??58KD?r?c???e?;?????? ?????C(??3),???F?3??pGEX4T1?3?B?<???????j ?z?c????GST?4?p???.(?:???????F28KD)?GCjaA (?:???????F30KD)???????9????,?w???????????? ?:?????G?????:???H?f???C??ke 5.???????????4????(??5) M:???:????????Marker;1:?6????;2:?????G????;3:?????G?j?o;4:?? ???G????;5:?.?y????1;6:?.?y????2 ??5????????rGSTCJAA?????????4SDSPAGE???? ?N??????????????,?????????m??????30min,?N ???????????G???j?o??,?3?J?4??Glutathione Sepharose 4B ???9????????,?3PH8.8,20mMGSH???. ?y???.?y????????,?310%???:???????YSDSPAGE ?b??,?3R250???????????y?????y???G?:???????? ???????4????ke ???? ?????f?????f???????????????????????E?m?; ??,?~?f?????%??ke???t,???t???????X?????G,???? ?????4???E?????c????50%~100%???6???F?C???? ?f????ke?????V?m?)?????:?????;?????????f????ke ???B?????????m??????,?????P?????????4?????;?Y ?F?u??????,???n???????c50?????????P???? [2] ke?? ???????????f?????;???????????K?Y?B?f???F?????? ?m??kp???????????????,????????ke 1972???????????????;???1?(?)?:???????f?? ??,???????X???6?????????F?????f?????????????? ???F?N,???T???X?????G?????f?????P???G?B?????? ?????? [3] ke?????f???????P???????????????????f ?????;???????????P???G????????????????,?????? ?????P???????????K?Y?f???????P???????<??ke?? 1921??Monsieur?u?????????????????z?%?????? (BCG)????,???????X?????3?3?????????????P???? ?P???)?????????3ke?u?????????o???????J???????? ??????,???????????????????F?Jke ???????????????c???9?e???????;???????:???~ ?R?????????????X??,???????????????4?????????? ?F?f?i?c?????????? [4] ke?????E?m?;?????????;?3 ?????;?B?B?????z???3,?????E?v?;?????????;?3?? ???;?B?B???????????j,?A?????f??????j?B???? ?4???j,?f?????c???????;?????u?<??ke?? 83?T?G?????????V??2011???(19???(2?P CaA 2008 Stuart?’ [5] ???3?????????3???????f??????CjaA?f ??????????,?X?????????????f?????w?o???5 [6] ke CjaA(Cj0982c:30kDa)??CjaC(Cj0734c:28kDa) ?G???z?P????ABC???????????c?A???;,CjaA???c ?????????;???,?A?w?o?????6?????c?????????;,?X CjaA?GCjaC???c?????A???;,????CjaA?M?F???c?? ?H???????i?c????ke?3Agnieszka?’ [7] ???????)???? ?t???cCjaA???????????????????????,????,???F?? ?F?????P?????f???????e?;IgG???????????:???; IgA????,?3?????;?????)???????????f???????P??ke CjaA?f?????f?????????i?c?????<??,?(?????? ?:???<?????f???k???????f????CjaA?????9????,?F ???????????????????????X?N???a???w,?????????? ?????????????f?????P????,???T???????P???G???? ?????????????P??ke ???n???8 [1]???/??,????,?l???/,?’.?????f???????t?H?????7???9?????? ?t?????S???X??.?T?G?????????V??,1994;2:6869. 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(P<0.01),ICACI?????????4?????c?B???????4????????VBCI??(P<0.05)keICACI???????????????c ?????4???????????????4?????c???4????,VBCI?????????????c???????4???????????????4???c???????4?? ??(<5)ke???c?????????Z???4?????G????kp???w??kp???w??kp???@??kp???l???c??(TG)kp???J??????(T)kp?? ?????????????e???J?????X?????]??????(YSN) ???o???????2,?????e???J?????????? 84?T?G?????????V??2011???(19???(2?P P0.0C :200801 :274000