Hasil Pencarian  ::  Simpan CSV :: Kembali

"To test effect one point mutation one the first initiation codon of Chiken Anemia Virus (CAV) open reading frame-3 (ORF-3), an opoptin knocked out expressor plasmid pCLS-VP3(-) and a CAV opoptin knocket out plasmid pCAV/Ap(-) were constructed. In both plasmids, the first ATG in COS-1 cells tranfectedwith pCLS-VP3(-) using western blotting and immunofluorescence type. After released from pCAV/Ap(-), the complete genome of CAV/Ap(-) was ligated to form the replicative form. the oppotin prodective was completely abolished in MDCC-MSB1 cells transfected with replicative form of CAV/Ap(-). The opoptin production was fully regained after a reverse mutation into CAV/Ap(-)RM. These data shows the first evidence that mutation of the first ATG of ORF-3 into ACG could completely abolish the production of apoptin"

"To test the effect of one point mutation on the first initiation codon of Chicken Anemia Virus (CAV) open reading frame-3 (ORF-3), an apoptin knocked out expressor plasmid pCLS-VP3(-) and a CAV apoptin knocket out plasmid pCAV/Ap(-) were constructed. In both plasmids, the first ATG in ORF-3 was mutated into ACG by a reversed long PCR. No apoptin detected in COS-1 cells transfected with pCLS-VP3(-) using western blotting and immunofluorescence assay, while apoptin was detected in COS-1 cells transfected with pCLS wild type. After released from pCAV/Ap(-), the complete genome of CAV/Ap(-) was ligated to form the replicative form. The apoptin production was completely abolished in MDCC-MSB1 cells transpected with replicate form of CAV/Ap(-). The apoptin production was fully regained after a reverse mutation into CAV/Ap(-)RM. These data shows the first evidence that mutation of the first ATG of ORF-3 into ACG could completely abolish the production of apoptin."

"Kanker merupakan gangguan kesehatan yang menjadi sebuah masalah besar di dunia. Kanker adalah penyakit akibat pertumbuhan tidak normal dari sel-sel jaringan tubuh yang berubah menjadi sel kanker. Apoptin diketahui memiliki kemampuan untuk memicu apoptosis di sel kanker secara in vitro maupun in vivo, tetapi tidak pada sel normal. Apoptin merupakan protein dari Chicken Anemia Virus (CAV) yang pertama kali diperkenalkan di Jepang pada 1974. Produksi Apoptin dapat dilakukan pada inang Eschericia coli dengan memindahkan gen Apoptin melalui vektor plasmid pET9a. Gen Apoptin yang digunakan telah dimodifikasi untuk meningkatkan efisiensi dan kemudahan dalam proses purifikasinya dengan penambahan beberapa tag dan situs pemotongan Thrombin. Purifikasi dilakukan menggunakan kromatografi afinitas ion logam (IMAC) nikel. Apoptin termodifikasi dengan HlyA-tag, (His)6-tag, (Arg)8-tag dan situs proteolitik thrombin berhasil diekpsresikan dan dipurifikasi di dalam E.coli DH5α dan BL21 dengan analisis SDS-PAGE. Optimasi ekspresi dilakukan dengan variasi strain E. coli membuktikan BL21 Codon Plus merupakan inang paling baik, konsentrasi IPTG lebih optimal pada 1 mM dibandingkan 0.4 mM, dan pengaruh suhu antara 28o dan 37o tidak signifikan. Binding Buffer dan Elution Buffer, paling baik dilakukan dengan komposisi: 20 mM sodium phosphate, 500 mM NaCl, dan 40 mM (binding) / 500 mM (elution) imidazole, pada pH 7.4.

---

Cancer is a health problem that is becoming a big problem in the world. Cancer is a disease caused by abnormal growth of tissue cells of the body that turn into cancer cells. Apoptin known to have the ability to trigger apoptosis in cancer cells in vitro and in vivo, but not in normal cells. Apoptin is a protein of Chicken Anemia Virus (CAV), which was first introduced in Japan in 1974. The production of Apoptin can be performed on the host Escherichia coli with gene transfer vector plasmid pET9a. Apoptin gene used has been modified to improve the efficiency and ease of purification process with the addition of a few tags and Thrombin proteolytic site.

Purification is done using ionic metal affinity chromatography (IMAC) nickel. Apoptin modified with HlyA-tag, (His)6-tag, (Arg)8-tag and thrombin proteolytic sites has been expressed and purified in E. coli DH5α and BL21 by SDS-PAGE analysis. Optimization conducted with several variation, expression in E. coli strain BL21 Codon Plus proved most optimum host, IPTG concentration at 1 mM given better expression than 0.4 mM, and the effect of temperature between 28o and 37o are insignificant. Binding buffer and the elution buffer, is best done with the composition: 20 mM sodium phosphate, 500 mM NaCl, and 40 mM (binding) or 500 mM (elution) imidazole in pH 7.4."

"Menurut data statistik WHO, kanker merupakan salah satu penyebab kematian terbesar di dunia, dengan 8,2 juta kematian dari 14 juta kasus kanker terhitung di tahun 2012. Lebih spesifik untuk wilayah Asia Tenggara, WHO mencatat 1,72 juta kasus dengan 1,17 juta kematian. Apoptin banyak diteliti sebagai protein dengan potensi terapi kanker, karena memiliki kemampuan menginduksi apoptosis di sel kanker secara spesifik. Potensi apoptin mendorong penelitian untuk apoptin dengan kemampuan penetrasi baik dan produksi dalam skala lebih besar. Dalam penelitian ini, telah dilakukan modifikasi apoptin dari chicken anemia virus dengan menggunakan affinity tag (His)6 , tag (Arg)8 untuk peningkatan penetrasi dan (HlyA) untuk sekresi apoptin keluar dari sel inang Escherichia coli ke media pertumbuhan sel secara genetik. Penambahan affinity tag (His)6 memungkinkan apoptin dipurifikasi dalam 1 langkah menggunakan kolom kromatografi nikel. Pelepasan (His)6 dan HlyA tag dirancang untuk dilakukan dengan menggunakan situs proteolitik thrombin. Protein diekspresikan dalam sel Escherichia coli strain BL21 pLysS, dan BL21 CodonPlus pada suhu 37oC dan 28oC. Analisis dilakukan dengan SDS PAGE memberikan hasil bahwa protein terekspresi dengan ukuran antara 20-25 kDa. Konfirmasi protein dilakukan dengan purifikasi kromatografi afinitas Nikel.

---

Based on WHO statistica data, cancer is one of the deadliest disease in the world, with 8,2 millions of deaths out of 14 million of cases in 2012. More specifically in South East Asia, WHO data showed 1,72 million of cases with 1,17 million of deaths. Apoptin intensively studied for its potential as a therapeutic protein for cancer treatment, since it able to induce apoptosis in cancer cells specifically. The apoptin potential drives researcher to produce apoptin in larger scale. In this research, chicken anemia virus apoptin have been modified by adding (His)6 tag, (Arg)8 tag and HlyA tag for purification, penetration and secretion from Escherichia coli to the growth media proposed. The addition of (His)6 tag enable apoptin to be purified in 1 step using nickel chromatography column. Removal of (His)6 tag and HlyA tag designed using specific thrombin proteolytic site. Protein expressed in Escherichia coli strain BL21 pLysS, and BL21 CodonPlus in temperature of 37oC and 28oC. Analysis done by SDS PAGE shows that the protein expressed with size between 20-25 kDa. Further confirmation done by Nickel affinity chromatography."

"Protein apoptin dari virus anemia ayam telah diteliti memiliki potensi yang baik sebagai pendeteksi dini sel kanker. Keberhasilan produksi apoptin yang tidak lagi berupa badan inklusi telah memberikan harapan lebih besar untuk melakukan optimasi produksi protein apoptin rekombinan ini. Sel rekombinan apoptin yang berhasil diproduksi dalam skala besar dengan menggunakan inang Bacillus subtilis 168 pOXGW-apop-2His8Arg, pOGW-apop-12His dalam berbagai variasi kondisi kultivasi (konsentrasi substrat penginduksi, laju aerasi, dan laju agitasi) kemudian dipurifikasi menggunakan metode IMAC dalam kolom afinitas (HisTrap FF 5 mL) yang berisi ion logam transisi Ni2+ dengan menggunakan instrumen AKTA Prime Plus. Secara rata-rata, hasil purifikasi menunjukkan bahwa elusi apoptin rekombinan terjadi ketika nilai konduktivitas berada pada angka 15,85 mS/cm, dengan konsentrasi imidazole berada pada kisaran nilai 76% - 88% (384,8-442,4 mM), yang terlihat dari grafik gradien elusi. Pengukuran konsentrasi protein apoptin dengan menggunakan metode Bradford menujukkan bahwa konsentrasi terbesar diperoleh pada sampel dengan sistem agitasi 250 rpm dan laju aerasi 0,5 Nl/menit, dengan besar konsentrasi 0,0507 mg/ml. Hasil purifikasi berhasil dideteksi menggunakan SDS-PAGE 12% dengan hasil pita protein terlihat di area 15 kDa dan 58,5 kDa untuk semua sampel.

---

Apoptin has been known to be having a great potency for cancer detection. The success of apoptin production which is not in inclusion body form anymore has given a bigger hope to optimize its production. Recombinant apoptin cells which was succesful to be cultivated in large scale using Bacillus subtilis 168 pOXGW-apop-12His8Arg, pOGW-apop-12His vector in various cultivation condition (aeration rate, agitation rate) then purified using IMAC method in Ni2+-loaded affinity column (HisTrap FF 5ml) and proceeded in AKTA Prime Plus instrument. Averagely, purifcation result showed that the elution of apoptin recombinant protein happened when the conductivity value at 15,85 mS/cm, with imidazole concentration lied around 76%-88% (384,8-442,4 mM), which could be seen from elusion gradient curve. The measurement of apoptin protein concentration using Bradford method showed that the biggest concentration was obtained from the sample with agitation rate 250 rpm and aeration rate 0,5 Nl/min, and the value is 0,0507 mg/ml. Purification yield was succesfully detected using SDS-PAGE 12%, with protein band was seen on 15 kDa and 58,5 kDa area for all samples."

"[Ebola adalah penyakit endemik yang telah terjadi selama tiga puluhan tahun setelah pertama kali ditemukan di daerah Afrika Barat pada tahun 1976. Penyakit ini disebabkan oleh virus ebola (EBOV) yang adalah salah satu bagian dari famili Filovoridae atau filovirus. Kasus ebola tidak dilaporkan secara intens, karena sifatnya yang endemik, namun tidak dapat dipungkiri, penyakit ini tetap memakan korban. Beberapa tahun terakhir, wabah ini merebak dan kembali menjadi pebincangan hangat. Penyebaran tersebut dikhawatirkan terjadi akibat adanya mutasi virus. Beberapa bagian lain mengkhawatirkan penggunaan virus tersebut dalam tindakan bioterorisme. Perkembangan virus dipengaruhi juga oleh proses replikasi, sehingga replikasi menjadi salah satu faktor penentu terjadinya penyakit. Pada ebola, replikasi dipengaruhi oleh protein VP35. Ilmu bioinformatika dianggap mampu menjadi salah satu cara menganalisis mutasi protein tersebut secara in silico, sehingga diharapkan para ilmuwan mampu menemukan desain inhibitor potensial untuk melawan ebola secara in vivo. Secara in silico telah diketahui perubahan yang terjadi pada VP35 virus ebola saat ini dibandingkan dengan yang ditemukan pertama kali pada tahun 1976. Perbedaan tersebut terjadi dalam hal susunan residu asam amino penyusun protein, terbukti dengan analisis pohon filogenetik, dan analisis lanjutan.;Ebola is an endemic disease which has been happened about thirty years after its first discovered in West Africa at 1976. It caused by ebolavirus (EBOV), member of Filoviridae. The ebola cases were not intensely reported, because it’s endemic disease, but the it still causes many deaths. Lately, ebola has been the headline, since its outbreak in West Africa, March 2014, and caused more than 50% mortality. Replication is one of the virulence factor. In ebolavirus, VP35 plays role in replication system, so the in silico study of VP35 ebolavirus is required, before the in vivo. Using bioinformatics tools, the differences between the 1976’s ebolavirus and 2014’s ebolavirus has been discovered. The differences are analyzed using phylogenetic tree, and advanced analyzing. ;Ebola is an endemic disease which has been happened about thirty years after its first discovered in West Africa at 1976. It caused by ebolavirus (EBOV), member of Filoviridae. The ebola cases were not intensely reported, because it’s endemic disease, but the it still causes many deaths. Lately, ebola has been the headline, since its outbreak in West Africa, March 2014, and caused more than 50% mortality. Replication is one of the virulence factor. In ebolavirus, VP35 plays role in replication system, so the in silico study of VP35 ebolavirus is required, before the in vivo. Using bioinformatics tools, the differences between the 1976’s ebolavirus and 2014’s ebolavirus has been discovered. The differences are analyzed using phylogenetic tree, and advanced analyzing. , Ebola is an endemic disease which has been happened about thirty years after its first discovered in West Africa at 1976. It caused by ebolavirus (EBOV), member of Filoviridae. The ebola cases were not intensely reported, because it’s endemic disease, but the it still causes many deaths. Lately, ebola has been the headline, since its outbreak in West Africa, March 2014, and caused more than 50% mortality. Replication is one of the virulence factor. In ebolavirus, VP35 plays role in replication system, so the in silico study of VP35 ebolavirus is required, before the in vivo. Using bioinformatics tools, the differences between the 1976’s ebolavirus and 2014’s ebolavirus has been discovered. The differences are analyzed using phylogenetic tree, and advanced analyzing. ]"