结核杆菌hsp基因缺失突变株的构建及鉴定-construction and identification of hsp gene deletion mutant of mycobacterium tuberculosis.docxVIP

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结核杆菌hsp基因缺失突变株的构建及鉴定-construction and identification of hsp gene deletion mutant of mycobacterium tuberculosis.docx

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结核杆菌hsp基因缺失突变株的构建及鉴定-construction and identification of hsp gene deletion mutant of mycobacterium tuberculosis

AbstractObjective:ToconstructandidentifythestrainsofMycobacteriumtuberculosisH37RvandH37RaandMycobacteriumbovisBacilleCalmette-Guérin(BCG)withHsp16.3geneknock-out.ItprovidesidealfeasibletoolforthestudiesofHsp16.3genefunction.Methods:MycobacteriumtuberculosisstrainsH37RvandH37RaandMycobacteriumbovisBCGstrainwereculturedinvitro,andtheH37RvgenomeDNAwasextracted.InordertoobtainagenetargetingvectorpKO-Hsp16.3,twofragments(5’-Hsp16.3,3’-Hsp16.3)flankingtheHsp16.3regionwereamplifiedbyPCRandinsertedintopKOplasmid,andtherecombinantpKO-Hsp16.3plasmidwasobtained,whichwasfurtheridentifiedbypolymerasechainreaction(PCR),digestedbydoublerestrictionenzymesandsequencing.Respectively,geneexchangetookplacewithinthegenomesofMycobacteriumtuberculosisH37RvandH37RaandMycobacteriumbovisBCGafterpKO-Hsp16.3plasmidweretransformedintoMycobacteriumtuberculosisH37RvandMycobacteriumtuberculosisH37RaandMycobacteriumbovisBCG,thestrainsofMycobacteriumtuberculosisH37RvwithHsp16.3geneknock-outandMycobacteriumtuberculosisH37RawithHsp16.3geneknock-outandMycobacteriumbovisBCGwithHsp16.3geneknock-outwereselected.Results:1.ThefragmentsflankingtheHsp16.3regionwereamplifiedandwereidentifiedbysequencing.ThetargetingvectorforthegeneencodingHsp16.3ofMycobacteriumtuberculosiswasconstructed,andsequencingverifiedthevectorscontainingtargetedgeneinspecificsites.Thetargetstrainswerethatofthepositivestrainbytwosteppolymerasechainreaction(PCR)andonestepsucrosecounterselection,withoutgrowthinculturemediawithhygomycin.Conclusions:ThestrainsofMycobacteriumtuberculosisH37RvwithHsp16.3geneknock-outandMycobacteriumtuberculosisH37RawithHsp16.3geneknock-outandMycobacteriumbovisBCGwithHsp16.3geneknock-outwereconstructedsuccessfully.Keywords:Geneknock-out,genetargetingvector,Hsp16.3,Mycobacteriumtuberculosis,electroporationTypeofThesis:A(BasicResearch)缩略词表英文缩写英文全称中文译名TBtuberculosis结核病MTBMycobacteriumtuberculosis结核分枝杆菌BCGMycobacteriumbovisBacilleCalmette-Guérin卡介苗MDR-TBmutipledrugsresistantBacillustuberculosis耐多药结核病sHSPssmallheat