IDENTIFIKASI MUTASI PADA DAERAH DNA POLIMERASE DAN HBsAg VIRUS HEPATITIS B

Infeksi virus hepatitis B (HBV) menyebabkan hepatitis akut dan kronis, dengan angka kematian 1,2 juta per tahun di seluruh dunia. Antivirus analog nukleosida dan vaksinHBV dapat menekan perkembangan infeksi HBV namun penggunaan untuk terapi jangkapanjang dilaporkan menginduksi terjadinya mutasi. Mutasi yang menyebabkan timbulmutan resisten-antivirus dan mutan lolos-vaksin menjadi kendala utama dalam pengobatandan pencegahan infeksi HBV. Telah dilakukan penelitian untuk mengidentifikasi mutasipada gen pengkode reverse transcriptase (RT) DNA polimerase dan HBsAg virus hepatitisB. Penelitian menggunakan 24 sampel cetakan HBV yang berasal dari penderita hepatitisB dari Medan (3), Jakarta (10), Bandung (9), Yogyakarta (1), dan Surabaya (1). Metodeyang dilakukan adalah amplifikasi fragmen gen pengkode DNA polimerase dan HBsAg,konfirmasi produk PCR dengan elektroforesis gel agarosa, pemurnian produk PCR dengan GFX column kit, penentuan urutan nukleotida, dan analisis hasil penentuan urutan nukleotida. Hasil penelitian menunjukkan sampel 12273 dari Jakarta mengalami mutasi di daerah gen pengkode RT DNA polimerase. Mutasi tersebut menyebabkan substitusi asam amino M475L, V519L, L526M, dan M550V. Sampel 12273 juga mengalami mutasi pada gen pengkode HBsAg yang menyebabkan substitusi asam amino M120L, V164L, L171M, dan M195V. Mutasi tersebut terjadi di daerah yang tumpang tindih dengan gen pengkode DNA polimerase, dan di luar determinan a. Mutan diklasifikasikan sebagai mutan resistenantivirus dengan substitusi asam amino ganda L526M dan M550V, dan tidak ditemukan mutasi pada daerah DNA pengkode determinan a.Kata kunci : HBV, RT DNA polimerase, HBsAg, Mutas

Overproduction, Purification and Refolding of codon-optimized Hepatitis B Virus X Protein Subgenotype B3 in Escherichia coli BL21(DE3)

Hepatitis B virus (HBV) infects human and causes chronic liver infection, leading to liver cirrhosis and hepatocellular carcinoma. HBV X (Hbx) protein is known to interact with tumor suppressor protein p53 and block its translocation into the nucleus. This study outlines the overproduction of Hbx protein from HBV subgenotype B3 in Escherichia coli BL21(DE3), including its purification and refolding. The gene encoding Hbx was first codon-optimized and inserted into pET16b. The recombinant plasmid was then transformed into E. coli BL21(DE3) as an expression host. Optimization of Hbx expression was performed with variation of IPTG concentration and overproduction temperature. The results showed that Hbx protein was optimally induced by 0.075 mM IPTG and overproduction of Hbx at 17, 25, and 37°C exhibited no difference in protein level and location. The optimal refolding of Hbx was obtained using 0.1 M arginine prior to elution from Nickel column using 100 mM imidazole and 0.25 M arginine. Hbx migrates differently in SDS-PAGE reducing and non-reducing, while the melting curve pattern in TSA analysis changed after the refolding step. Essentially, this purified Hbx protein could potentially be used for interaction study with p53 and the inhibitor candidate of the protein

Β-LACTAMASES INHIBITOR-PRODUCING SOIL BACTERIA FOR AMPICILLIN-RESISTANT UROPATHOGENIC ESCHERICHIA COLI ISOLATE

Objective: The goals of this investigation were to identify the species of the producers and ascertain the dose-dependent effect of extracellular products of Indonesian bacteria that generate β-lactamases inhibitors. Methods: An agar diffusion technique for the lactamase inhibitor activity assay was performed. Observation of bacteria using phenotypic analysis was performed by observing colony color and cell shape morphology, biochemical assays and a series of carbohydrate fermentation tests. Bacterial identification was performed by comparing the nucleotide sequence of the 16S rDNA gene of target bacteria with available nucleotide sequences in Gene Library (NCBI). Combining data from phenotypic and genotypic analyses allowed for the identification of the producers. Results: According to our findings, none of the bacteria's extracellular products, which contain β-lactamase inhibitors in a range of concentrations, showed a discernible impact on the values of the inhibition zone. The producers are Aeromonas popoffii, Alcaligenes faecalis, Streptomyces brasiliensis, Staphylococcus equorum, Pseudomonas putida, Pseudomonas fluorescens, Salmonella typhi, Enterobacter hormaechei, Serratia marcescens and Enterobacter sp. The highest potency of β-lactamase inhibitor was provided by the extracellular product of VR3 isolate bacteria which was identified as Serratia marcescens. Conclusion: In conclusion, this study clearly showed that our isolated bacteria have the potential to be further investigated in order to maximize the recovery of β-lactamase inhibitor compounds

The light subunit of mushroom Agaricus bisporus tyrosinase:Its biological characteristics and implications

The light subunit of mushroom Agaricus bisporus tyrosinase (LSMT) is a protein of unknown function that was discovered serendipitously during the elucidation of the crystal structure of the enzyme. The protein is non-immunogenic and can penetrate the intestinal epithelial cell barrier, and thus, similar to its structural homologue HA-33 from Clostridium botulinum, may be potentially absorbable by the intestine. LSMT also shares high structural homology with the ricin-B-like lectin from the mushroom Clitocybe nebularis (CNL), which has been shown to display biological activity against leukemic cancer cells and dendritic cells. Therefore, we evaluated the biological activity of LSMT. An in vitro assay suggested that LSMT presentation to most of the cancer cell lines studied has a negligible effect on their proliferation. However, inhibition of cell growth and a slight stimulation of cell proliferation were observed with breast cancer and macrophage cells, respectively. LSMT appeared to be relatively resistant against proteolysis by trypsin and papain, but not bromelain. Challenges with gastric and intestinal juice suggested that the protein is resistant to gastrointestinal tract conditions. This is the first report on the biological characteristics and implication of LSMT. (C) 2017 Elsevier B.V. All rights reserved

AMPLIFIKASI DAN SEKUENSING GEN ESAT-6 DARI Mycobacterium tuberculosis SEBAGAI KANDIDAT VAKSIN TUBERKULOSIS

Penelitian ini bertujuan untuk mengamplifikasi dan mengurutkan urutan nukleotida gen pengkode ESAT-6 Mycobacterium tuberculosis beberapa isolat klinis dari beberapa daerah di indonesia. ESAT-6 merupakan antigen untuk kandidat vaksin tuberberkulosisSampai saat ini, vaksin BCG untuk mencegah tuberkulosis tidak berhasil, karena tetap tingginya penderita tuberkulosis di dunia. Demikian pula di Indonesia, walaupun hampir semua penduduk Indonesia telah divaksinasi dengan BCG, namun jumlah penderita tuberkulosis selalu meningkat dan menjadikan Indonesia pada peringkat ketiga tertinggi di dunia setelah India dan Cina. Oleh karena itu, diperlukan pengganti vaksin BCG yang efektif sehingga pemberantasan tuberkulosis dapat dilakukan.\ud Salah satu antigen yang banyak diteliti sebagai kandidat vaksin tuberkulosis adalah early secreted antigen target yang berbobot molekul 6 kDa (ESAT-6). Protein ini disekresikan oleh M. tuberculosis pada fase awal dari pertumbuhannya, dan telah dibuktikan dapat menginduksi produksi interferon gamma (INF???) oleh sel T CD8. INF??? ini dapat mengaktivasi makrofag yang terinfeksi M. tuberculosis untuk membunuh bakteri tersebut. \ud Tujuan penelitian ini adalah untuk mengamplifikasi gen ESAT-6 M.tuberculosis dan mengurutkan urutan nukleotidanya dengan sekuensing. Tahapan penelitian yang digunakan adalah pengumpulan sampel dari beberapa daerah di Indonesia, mengisolasi DNA genom M. tuberculosis dengan metode Boom, merancang primer ESAT-6, mengamplifikasi dengan PCR dan analisis data sekuensing dilakukan dengan BLAST. \ud Hasil penelitian diperoleh gen pengkode ESAT-6 M. tuberculosis dengan ukuran 285 bp dan dari analis sekuensing menunjukkan bahwa urutan nukleotida dan urutan asam amino yang dideduksi memiliki tingkat konservasi yang tinggi, yaitu 95 ??? 100%.

A novel immune-tolerable and permeable lectin-like protein from mushroom Agaricus bisporus

A lectin like protein designated as LSMT is recently discovered in Agaricus bisporus. The protein adopts very similar structure to Ricin-B like lectin from Clitocybe nebularis (CNL) and HA-33 from Clostridium botulinum (HA-33), which both recognize sugar molecules that decorate the surface of the epithelial cells of the intestine. A preliminary study in silico pointed out potential capability of LSMT to perform such biological activity. Following that hypothesis, we demonstrated that LSMT is indeed capable of penetrating out from a dialysis tube of the mice intestine origin. Furthermore, the protein appeared not to evoke the immune response upon introduction into mice, unlike its structural homologs. This is the first report on the biological implication of LSMT that might lead to its application. (C) 2016 Elsevier Inc. All rights reserved