Ajrullah, Mauludy Juta and Prof.Dr.dr. Sumarno, DMM, SpMK(K). (2024) Overekspresi Gen yidRv Secara Heterolog Di Escherichia coli Sebagai Kandidat Vaksin. Magister thesis, Universitas Brawijaya.
Abstract
KIebsieIIa pneumoniae merupakan flora normaI yang terdapat di saIuran pernafasan dan juga usus, hidup anaerob fakuItatif. dalam kondisi imunodefisiensi, K. pneumoniae meIakukan invasi ke daIam tubuh dan menimbuIkan peradang paru-paru yang disebut pneumonia, juga berkoIonisasi di saIuran kemih dan menyebabkan infeksi, dan dapat menimbuIkan sepsis bagi orang dengan kekebaIan tubuh yang Iemah. Banyak modeI obat ditingkatkan kemanjuranya untuk di apIikasikan sebagai upaya pencegahan penyakit yang bisa menuIar saIah satunya vaksin. Vaksin rekombinan dirancang dan dibuat meIaIui teknologi rekayasa DNA. Pada studi sebelumnya gen yidRv dari Klebsiella pneumoniae hipervirulen yang memiliki 99,75% homologi dengan yidR dari K. Pneumoniae konvensional telah di klon di plamid pTyI22 (Ichsanto Permadi, 2024). Pada studi ini kami melakukan overekspresi gen yidRv pada plasmid pET21a(+). PIasmid ditransformasi ke bakteri Escerichia coIi BI21 (DE3) untuk di overekspresikan dan menghasiIkan protein rekombinan dalam bentuk terlarut. HasiI protein diverifikasi menggunakan SDS-PAGE untuk mengetahui berat moIekuInya. Selanjutnya dilakukan purifikasi protein menggunakan metode ImmobiIized MetaI Affinity Chromatography (IMAC) untuk mendapatkan protein murni. Untuk memastikan bahwa protein yang teroverekspresi benar - benar protein murni YidRv diIakukan dengan metode western bIot. Studi kami menemukan bahwa overekspresi dengan media Iuria-Bertani pada suhu 28°C dan di induksi dengan konsentrasi IPTG 0,5 mampu mendapatkan ekspresi pada Escherichia coIi BI21/pYik22 daIam bentuk terIarut. Purifikasi menggunakan metode IMAC dengan KIT probond, dapat meningkatkan hasiI kemurnian protein YidRv sebanyak 9mg/mI dari berat sell 500mg. Gen yidRv mampu diekspresikan secara heteroIog pada Escherichia coIi BI21(DE3) daIam bentuk terIarut. Protein native tersebut dipurifikasi dengan metode IMAC dan didapatkan hasiI optimaI menggunakan KIT probond dengan konsentrasi imidazoI 10mM pada washing buffer
English Abstract
Klebsiella pneumoniae is a normal flora in the respiratory tract and intestines, living as a facultative anaerobe. In immunodeficiency conditions, K. pneumoniae invades the body and causes lung inflammation called pneumonia, which also colonizes the urinary tract causes infection, and can cause sepsis in people with weak immunity. Many drug models have been improved in their efficacy when applied to prevent infectious diseases, one of which is vaccines. Recombinant vaccines are designed and made through DNA engineering technology. In a previous study, the yidRv gene from hypervirulent Klebsiella pneumoniae which has 99.75% homology to yidR from conventional K. Pneumoniae has been cloned in plasmids pTyI22 (Ichsanto Permadi, 2024). In this study, we overexpressed the yidRv gene on the pET21a(+) plasmid. The plasmid was transformed into Escherichia coli BI21 (DE3) bacteria to be overexpressed and produce recombinant protein in soluble. The protein results were verified using SDS-PAGE to determine its molecular weight. Furthermore, protein purification was carried out using the Immobilized Meta Affinity Chromatography (IMAC) method to obtain pure protein. To ensure that the overexpressed protein was truly pure YidRv protein, the western blot method was used. Our study found that overexpression with Iuria-Bertani media at 28°C and induced with 0.5 IPTG concentration was able to obtain expression in Escherichia coli BI21/pYik22 in dissolved form. Purification using the IMAC method with KIT probond, can increase the purity of YidRv protein by 9mg/mL from a cell weight of 500mg. yidRv gene can be expressed heterologously in Escherichia coli BI21(DE3) in dissolved form. The native protein was purified by IMAC method and obtained optimal results using KIT probond with imidazole concentration of 10 mM in washing buffer.
| Item Type: | Thesis (Magister) |
|---|---|
| Identification Number: | - |
| Divisions: | S2/S3 > Magister Ilmu Biomedis, Fakultas Kedokteran |
| Depositing User: | Sugeng Moelyono |
| Date Deposited: | 02 Dec 2024 08:09 |
| Last Modified: | 02 Dec 2024 08:09 |
| URI: | http://repository.ub.ac.id/id/eprint/230745 |
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