| Abstract | Penelitian ini bertujuan mengetahui apakah strategi yang digunakan dapat berhasil mengkloning danmengekspresikan sekuen DNA target (daerah DBL3x gen var2csa) pada inang E.coli. Strategi kloning dilakukanmelalui tahapan: perbanyakan sekuen DNA target melalui teknik PCR menggunakan primer yang didesain sendiri,peligasian sekuen DNA target pada vektor kloning pCR2.1ïÃÂâÂÂ-TOPOïÃÂâÂÂ, dan memperbanyaknya pada inang E.coliTOP10. Strategi ekspresi dilakukan melalui tahapan: perbanyakan sekuen DNA target melalui teknik PCRmenggunakan primer yang mengandung situs pengenalan enzim restriksi endonuklease tipe II: NcoI dan XhoI,pemotongan dengan enzim NcoI dan XhoI, peligasian pada vektor ekspresi pET-28a (+), dan pengekspresian padainang E. coli BL21 (DE3) dengan induser isopropyl-1-thio-ÃÂÃ
¸-D-galactoside (IPTG) 1 mM. Strategi kloningberhasil mendapatkan sekuen daerah DBL3x gen var2csa berukuran 948 pb. Strategi ekspresi berhasilmendapatkan protein DBL3x berukuran 38 kDa.Kata kunci: kloning, ekspresi, DBL3x, var2csa, Escherichia coliABSTRACTThe objective of this study is to see are the strategies approach that used successfully clone and expressDBL3x region of var2csa gene in E.coli host. The cloning strategies are amplifying DNA target through PCRtechnique using self design oligonucleotide primer, inserting into cloning vector pCR2.1ïÃÂâÂÂ-TOPOïÃÂâÂÂ, copying inE.coli TOP10 host. The expression strategies are amplifying DNA target through PCR using self design primerwith specific site for restriction enzyme NcoI and XhoI, digesting with NcoI and XhoI, inserting into pET-28a (+)expression vector, and expressing in E. coli BL21 (DE3) host by adding isopropyl-1-thio-ÃÂÃ
¸-D-galactoside (IPTG)1 mM as a inducer. This study successfully cloned DBL3x region of var2csa gene with 948 pb in size andsuccessfully expressed protein DBL3x with 38 kDa in size in E.coli system.Keywords: cloning, expression, DBL3x, var2csa, Escherichia coli |
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