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"Demam Dengue (DD) dan Demam Berdarah Dengue (DBD) adalah penyakit yang tersebar luas. Penelitian ini dilakukan untuk mengembangkan kandidat vaksin dengue nasional berbasis protein sub unit prM/E DEN-4. Protein prM/E adalah kompleks unik yang berperan penting dalam perakitan virus dan modulasi fusi. Penelitian telah dilakukan dengan metode Gateway cloning system untuk menklon gen prM/E dalam plasmid cloning pDONR221 kemudian dilakukan subkloning dan dipindahkan gen prM/E ke dalam plasmid eksperesi pET-55-DEST. Ekspresi protein prM/E dilakukan di dalam E.coli BL21 (DE3) dengan induksi Isoprophyl-β-D-thiogalactopyranoside (IPTG). Pendeteksian poliprotein Gag hasil ekspresi dilakukan dengan metode Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis (SDS-PAGE). Setelah protein prM/E berhasil dideteksi kemudian protein prM/E dipurifikasi dengan menggunakan metode immobilized metal affinity chromatography (IMAC) di bawah kondisi denaturasi. Hasil penelitian yaitu protein prM/E dapat diekspresikan dalam E.coli BL21 (DE3) dengan berat molekul ~75 kDa.

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Dengue Fever is an infectious disease caused by one of the four serotypes of dengue virus (DENV). Until now, no licensed vaccines or antivirus is available commercially. Because of that, this research was aimed to develop candidate vaccine dengue based on protein subunit pre-membrane and Envelope (prM/E). Protein prM/E is a unique complex which has important role in virus assemblyand host cell entry. The recombinant protein development was done using Gateway cloning system. This was used to clone the prM/E gene into pDONR221 plasmid. The cloned gene was then transferred into pET-55-DEST expression plasmid. Expression of protein prM/E was perfomed in E. coli BL21 (DE3) with inducer Isoprophyl-β-D-thiogalactopyranoside (IPTG). Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis (SDS-PAGE) method was used to detect the expressed prM/E protein. Upon detection of prM/E protein with SDS-PAGE, the recombinant protein was purified by using immobilized metal affinity chromatography (IMAC) method under denature condition. Using these methods, the prM/E protein was successfully expressed in E.coli BL21 (DE3) with a molecular weight ~75 kDa."

"Protein NS3 pada Dengue Virus DENV Serotipe 3 DENV-3 adalah protein nonstruktural yang memiliki berat molekul 72 kDa dan bertanggung jawab dalam siklus replikasi virus dengue. Protein tersebut dapat dijadikan kandidat vaksin rekombinan subunit penyakit Demam Berdarah DBD . Penelitian ini bertujuan untuk validasi hasil kloning gen NS3 DENV-3 ke vektor pYES2/CT sebelumnya, ekspresi dan purifikasi protein rekombinan dari sel Saccharomyces cerevisiae. Uji validitas dengan metode PCR dan analisa sekuensing DNA menunjukkan bahwa gen NS3 DENV-3 pada klon 2 dan 11 memiliki validitas yang tinggi terinsersi pada plasmid pYES2CT >90. Hasil ekspresi transforman Saccharomyces cerevisiae pYES2/CT dengan metode SDS PAGE dan Western Blot menunjukkan adanya pita spesifik berukuran 72 kDA pada sampel 2A dan 8B. Hasil purifikasi sampel yang sudah terverifikasi ekspresinya sampel 2A dengan mekanisme elusi gradien menunjukkan adanya protein spesifik yang terelusi dengan menggunakan elution buffer yang mengandung imidazol konsentrasi 350 mM.

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DENV 3 NS3 Protein is a non structural protein with molecular weight approximately around 72 kDA and responsible for replication cycle dengue virus. This protein could be a candidate for subunit recombinant vaccine of Dengue Haemorrhagic Fever DHF. The aims of this study were to validated the previous cloning result of DENV3 NS3 gene into pYES2 CT vector, expressed and purified the recombinant protein from Saccharomyces cerevisiae cells. The result of validity tests with PCR method and DNA sequencing showed NS3 gene in clone 2 and 11 had high validity were inserted on plasmid pYES2 CT 90. The result of the expression in transformant Saccharomyces cerevisiae pYES2 CT with SDS PAGE and Western Blot methods showed there was a specific band with size 72 kDa in clone 2A and 8B. The result of verified clone clone 2A with gradient elution mechanism showed there was a specific protein that was eluted by elution buffer which contained 350 mM imidazole."

"ABSTRAKGen NS1 merupakan gen penyandi protein NS1 (non-strukural 1) yang terdapat pada virus dengue. Protein NS1 diketahui memiliki potensi untuk dikembangkan sebagai bahan dasar kit diagnostik untuk penyakit demam berdarah dengue (DBD). Penelitian ini bertujuan untuk memperoleh protein rekombinan NS1 virus dengue untuk pengembangan kit diagnostik NS1. Penelitian ini meliputi proses kloning gen NS1 pada vektor ekspresi pYES2/CT, ekspresi pada Saccharomyces cerevisiae INVSc1 dan purifikasi protein rekombinan NS1 menggunakan HisPurTM Ni-NTA Magnetic Beads. Hasil penelitian menunjukkan sebanyak 485 koloni transforman hasil kloning ke dalam E. coli TOP10F? berhasil diseleksi pada medium yang mengandung 100 µg/ml. Analisis hasil PCR dan sekuensing menunjukkan bahwa gen NS1 yang berukuran 1056 pb berhasil terintegrasi ke dalam vektor ekspresi pYES2/CT. Analisis hasil SDS PAGE dan western blotting menunjukkan protein rekombinan NS1 berhasil diekspresikan pada Saccharomyces cerevisiae dengan ukuran sekitar 42?55 kDa. Analisis SDS PAGE untuk hasil purifikasi menunjukkan didapatkan protein yang terelusi dalam kondisi native dengan ukuran sekitar 42?55 kDa. Gen NS1 telah berhasil dikloning dan protein NS1 berhasil terekspresi serta terpurifikasi.

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ABSTRAKNS1 gene is a gene encoding NS1 (non-structural 1) protein dengue virus. Dengue NS1 protein (non-structural 1) is known as an important biomarker for early diagnosis of dengue hemorrhagic fever (DHF) disease. The research objective is to obtain NS1 recombinant protein dengue virus serotype 3 for development kit diagnostic NS1. Stages of the research include cloning NS1 gene Into pYES2/CT expression vector, expression in Saccharomyces cerevisiae INVSc1, and purification NS1 recombinant protein with HisPurTM Ni-NTA Magnetic Beads. A total of 485 colony transformants were selected on medium with ampicilin 100 µg/ml as cloning results in E. coli TOP 10F?. PCR and sequencing analysis showed that NS1 gene was successfully fused to vector pYES2/CT and showed NS1 size is 1056 bp. SDS PAGE and western blotting analysis showed a band of NS1 recombinant protein as expression results. Molecular weight of NS1 protein was approximately 42--55 kDa. SDS PAGE analysis showed a band of NS1 recombinant protein purified in native condition with a molecular weight approximately 42--55 kDa. NS1 gene was successfully cloned, can be also expressed and purified as protein NS1."

"[ABSTRAKDemam berdarah dengue (DBD) merupakan penyakit yang disebabkan karenainfeksi virus dengue (DENV), yang banyak ditemukan di Indonesia. Belum adaterapi yang spesifik dalam pengobatan DBD. Upaya pengembangan vaksindengue yang efektif sangat diperlukan. Pada penelitian ini dilakukan analisaimunogenisitas kandidat vaksin DNA prM-E dengue serotipe 2 (pUMD2.kl.20)dengan menganalisis sel T CD4, sel T CD8, IFN-γ dan TNF-α. pada sel U937 danPBMC secara in vitro, bertujuan untuk mengetahui respon imun ketika vaksindiinjeksikan ke dalam tubuh manusia. Dasar penelitian ini adalah sel U937ditransfeksi dengan pUMD2.kl.20 menggunakan lipofectamin. Sel U937 akanberperan sebagai APCs yang akan mengekspresikan protein prM-E DENV-2 danmempresentasikan protein tersebut melalui molekul MHC class I dan MHC classII kepada Peripheral Blood Mononuclear Cell (PBMC) manusia. Tahapan kerjayang dilakukan dalam penelitian ini terdiri dari: (a) kultur galur sel U937, (b)transfeksi sel U937 dengan pUMD2.kl.20, (c) transfeksi sel U937 dengan plasmids(pUMVC), (d) infeksi sel U937 dengan DENV-2 strain DS.18/09, (e) pewarnaandan pengamatan hasil pewarnaan menggunakan alat semi flowcytometri (TALI).pUMD2.kl.20 mengaktivasi sel T CD4 dan sel T CD8 untuk berploriferasi. Hasilpenelitian ini menunjukkan bahwa konsentrasi sel T CD8 lebih tinggi darikonsentrasi sel T CD4 dan konsentrasi sel positif yang mensekresikan IFNγ lebihtinggi dari konsentrasi sel positif yang mensekresikan TNFα. Kondisi optimal dariaktivasi sel T CD4 oleh pUMD2.kl.20 adalah 24 jam setelah penambahan PBMCsetelah transfeksi (8,53x10⁴sel/ml), untuk sel T CD8 adalah 24 jam setelahpenambahan PBMC setelah transfeksi (49,4x10⁴ sel/ml). Kondisi optimal dariaktivasi sel+ yang mensekresikan IFNγ oleh pUMD2.kl.20 adalah 24 jam setelahpenambahan PBMC 48 jam setelah transfeksi (9,86x10⁴ sel/ml), dan untuk sel+yang mensekresikan TNFα adalah 2 jam setelah penambahan PBMC 48 jamsetelah transfeksi (2,1x10⁴ sel/ml). Dari penelitian ini dapat disimpulkan bahwapUMD2.kl.20 bersifat imunogenik.

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ABSTRACTDengue hemorrhagic fever (DHF) is a disease caused by infection with denguevirus (DENV), which is found in Indonesia. There is no specific therapy in thetreatment of DHF. An effort to develop an effective dengue vaccine is needed. Inthis research, analysis of the immunogenicity of the DNA vaccine candidate prMEdengue serotype 2 (pUMD2.kl.20) by analyzing the CD4 T cells, CD8 T cells,IFN-γ and TNF-α in U937 cells and PBMC in vitro, aims to determine theimmune response when the vaccine is injected into the human body. This researchapproach is based on transfection of U937 cells with pUMD2.kl.20 usinglipofectamin. U937 cells acts as APCs which will express the protein prM-EDENV-2 and presenting these proteins through the MHC class I and MHC class IImolecule to the Peripheral Blood Mononuclear Cell (PBMC) of human body.Stages of the work in this study consisted of: (a) culture cell line U937, (b)transfection of U937 cells with pUMD2.kl.20, (c) transfection of U937 cells withplasmid (pUMVC), (d) infection of U937 cells with DENV-2 strains DS.18/09,(e) staining and observation results of staining using a semi-flow cytometri(TALI). pUMD2.kl.20 activate CD4 T cells and CD8 T cells to proliferate. CD8 Tcells concentration higher than the concentration of CD4 T cells and the secretionof INFγ-positive cells concentration higher than the concentration of the secretionof TNFα-positive cells. Optimal condition of CD4 T cells activation bypUMD2.kl.20 is 24 hours after the addition of PBMC after transfection (8,53x10⁴cells/ml), for CD8 T cells was 24 hours after the addition of PBMC aftertransfection (49,4x10⁴ cells/ml). Optimal conditions secretion of IFNγ-positivecells were activated by pUMD2.kl.20 is 24 hours after the addition of PBMC 48hours after transfection (9,86x10⁴ cells/ml), and for the secretion of TNFα-positive cells were activated by pUMD2.kl.20 is 2 hours after the addition ofPBMC 48 hours after transfection (2,1x10⁴ cells/ml). From this study it can beconcluded that the pUMD2.kl.20 immunogenic, Dengue hemorrhagic fever (DHF) is a disease caused by infection with denguevirus (DENV), which is found in Indonesia. There is no specific therapy in thetreatment of DHF. An effort to develop an effective dengue vaccine is needed. Inthis research, analysis of the immunogenicity of the DNA vaccine candidate prMEdengue serotype 2 (pUMD2.kl.20) by analyzing the CD4 T cells, CD8 T cells,IFN-γ and TNF-α in U937 cells and PBMC in vitro, aims to determine theimmune response when the vaccine is injected into the human body. This researchapproach is based on transfection of U937 cells with pUMD2.kl.20 usinglipofectamin. U937 cells acts as APCs which will express the protein prM-EDENV-2 and presenting these proteins through the MHC class I and MHC class IImolecule to the Peripheral Blood Mononuclear Cell (PBMC) of human body.Stages of the work in this study consisted of: (a) culture cell line U937, (b)transfection of U937 cells with pUMD2.kl.20, (c) transfection of U937 cells withplasmid (pUMVC), (d) infection of U937 cells with DENV-2 strains DS.18/09,(e) staining and observation results of staining using a semi-flow cytometri(TALI). pUMD2.kl.20 activate CD4 T cells and CD8 T cells to proliferate. CD8 Tcells concentration higher than the concentration of CD4 T cells and the secretionof INFγ-positive cells concentration higher than the concentration of the secretionof TNFα-positive cells. Optimal condition of CD4 T cells activation bypUMD2.kl.20 is 24 hours after the addition of PBMC after transfection (8,53x10⁴cells/ml), for CD8 T cells was 24 hours after the addition of PBMC aftertransfection (49,4x10⁴ cells/ml). Optimal conditions secretion of IFNγ-positivecells were activated by pUMD2.kl.20 is 24 hours after the addition of PBMC 48hours after transfection (9,86x10⁴ cells/ml), and for the secretion of TNFα-positive cells were activated by pUMD2.kl.20 is 2 hours after the addition ofPBMC 48 hours after transfection (2,1x10⁴ cells/ml). From this study it can beconcluded that the pUMD2.kl.20 immunogenic]"

"ABSTRAKPenyakit Demam Berdarah Dengue (DBD) di Indonesia paling dominandisebabkan oleh virus dengue (DENV) serotipe 3. Upaya pencegahan DBD dapatdilakukan melalui vaksinasi. Lembaga BPPT saat ini sedang mengembangkanvaksin DBD berbahan baku protein rekombinan NS2B-NS3. Protein inimerupakan salah satu protein non struktural penyusun genom DENV danmemiliki berat molekul sebesar 83 kDa. Penelitian ini bertujuan untuk melakukanisolasi dan purifikasi protein NS2B-NS3 DENV serotipe 3 dari sel transformanSaccharomyces cerevisae. Purifikasi protein NS2B-NS3 dilakukan dengan metodeHisPur Ni-NTA Magnetic Beads. Optimasi purifikasi dilakukan denganmeningkatkan konsentrasi imidazole sebagai pengikat protein dalam elution bufferdari 250 mM -- 500 mM. Validitas isolat protein dan protein hasil purifikasi diujisecara kualitatif dengan metode Sodium Dodecyl Sulfate-Polyacriamide GelElectrophoresis (SDS-PAGE), serta dikuantifikasi proteinnya dengan metodeBichinconinic Acid (BCA). Hasil penelitian menunjukkan bahwa protein NS2BNS3telah berhasil dipurifikasi secara optimal pada konsentrasi imidazole 300mM dengan metode HisPur Ni-NTA Magnetic Beads. Analisis hasil SDS-PAGEmenunjukkan bahwa terdapat pita spesifik berukuran 83 kDa pada lajur hasil elusidengan konsentrasi imidazole 300 mM dan berdasarkan hasil kuantifikasi proteindiperoleh persentase efektivitas purifikasi tertinggi, yaitu 16,38%.

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ABSTRACTPenyakit Demam Berdarah Dengue (DBD) di Indonesia paling dominandisebabkan oleh virus dengue (DENV) serotipe 3. Upaya pencegahan DBD dapatdilakukan melalui vaksinasi. Lembaga BPPT saat ini sedang mengembangkanvaksin DBD berbahan baku protein rekombinan NS2B-NS3. Protein inimerupakan salah satu protein non struktural penyusun genom DENV danmemiliki berat molekul sebesar 83 kDa. Penelitian ini bertujuan untuk melakukanisolasi dan purifikasi protein NS2B-NS3 DENV serotipe 3 dari sel transformanSaccharomyces cerevisae. Purifikasi protein NS2B-NS3 dilakukan dengan metodeHisPur Ni-NTA Magnetic Beads. Optimasi purifikasi dilakukan denganmeningkatkan konsentrasi imidazole sebagai pengikat protein dalam elution bufferdari 250 mM -- 500 mM. Validitas isolat protein dan protein hasil purifikasi diujisecara kualitatif dengan metode Sodium Dodecyl Sulfate-Polyacriamide GelElectrophoresis (SDS-PAGE), serta dikuantifikasi proteinnya dengan metodeBichinconinic Acid (BCA). Hasil penelitian menunjukkan bahwa protein NS2BNS3telah berhasil dipurifikasi secara optimal pada konsentrasi imidazole 300mM dengan metode HisPur Ni-NTA Magnetic Beads. Analisis hasil SDS-PAGEmenunjukkan bahwa terdapat pita spesifik berukuran 83 kDa pada lajur hasil elusidengan konsentrasi imidazole 300 mM dan berdasarkan hasil kuantifikasi proteindiperoleh persentase efektivitas purifikasi tertinggi, yaitu 16,38%."

"ABSTRAKInfeksi yang disebabkan oleh virus dengue telah banyak dilaporkan di negara tropis dan subtropis. Virus dengue terdiri dari 4 serotipe yaitu dengue 1-4. Hingga saat ini belum tersedia vaksin yang berlisensi untuk mencegah terjadinya infeksi dengue. Pada penelitian ini dikonstruksi vaksin DNA yang mengkode gen prM-E dan prM-E-NS1del virus dengue 2 strain Indonesia yang akan dijadikan sebagai kandidat vaksin dengue. Hasil penelitian berhasil mendapatkan 9 plasmid rekombinan pUMDE2 yang membawa gen sisipan prM-E dan telah dikonfirmasi dengan melakukan PCR koloni dan restriksi plasmid. Dari hasil sekuensing plasmid pUMDE2 koloni no. 11 ditemukan 19 mutasi asam amino pada gen prM-E, sepuluh mutasi pada gen prM dan sembilan mutasi pada gen E. Mutasi protein prM N29D dan N52K serta protein E V164I dan S390N terletak pada daerah epitop pengenalan sel B. Transfeksi plasmid pUMDE2 dilakukan pada sel Chinese Hamster Ovary (CHO)-K1 dan menunjukkan adanya ekspresi protein prM-E rekombinan berdasarkan uji imunostaining dan ELISA. Hasil ELISA menunjukkan bahwa protein ditemukan pada sel yang ditransfeksi. Sedangkan, plasmid rekombinan yang membawa gen prM-E-NS1del tidak berhasil dikonstruksi. Plasmid pUMDE2 dapat dikembangkan menjadi kandidat vaksin DNA.

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ABSTRACT

Infection by dengue virus were reported in tropical and subtropical area. Dengue virus (DENV) consist of 4 serotype, DENV-1 to DENV-4. There is no licensed vaccine available for dengue infection. In this research, we construct DNA vaccine encode prM-E and prM-E-NS1del genes of dengue virus serotype 2 for vaccine development. Nine recombinant plasmids that encode prM-E genes (pUMDE2), were successfully obtained. Recombinant plasmids were confirmed by PCR colony and restriction enzyme analysis. Colony of pUMDE2 no. 11 was sequenced and total 19 amino acid mutations were founds, 10 mutations in prM and 9 mutations in E protein. prM mutations N29D and N52K, E mutations of V164I and S390N were found in B cell epitopes. Transfection pUMDE2 plasmid was done to Chineese Hamster Ovary (CHO)-K1 and showed that recombinant protein prM-E was successfully expressed by immunostaining assay and ELISA. Results showed that the protein was mainly found in cell fraction. However, recombinant plasmid that encode prM-E-NS1del were failed to be constructed. pUMDE2 could be developed for vaccine candidate."

" ABSTRAK Dengue Hemorrhagic Fever (DHF) are viral diseases transmitted by mosquito vector spreads fastest in the world. Cause of dengue fever are RNA virus family Flaviviridae called dengue virus (DENV). DENV genome encodes three structural proteins, capsid (C), membrane proteins (prM), envelope protein (E) and seven nonstuktural protein NS1, NS2a, NS2b, NS3, NS4a, NS4B, and NS5. NS3 protein contains many epitopes that can be recognized by the humoral and cellular immune system. Therefore NS3 protein is a potential target for development of dengue vaccines. This study begins by sequencing NS3 gene DENV-4 IDS 96/10. Phylogenetic analysis and epitope analysis were done from the result of sequencing. Phylogenetic analysis showed IDS 96/10 are in one clade with strains isolated rom China (2010), Singapore (2010) and Thailand (2000). NS3 gene DENV-4 IDS 96/10 contained epitopes recognized by CD4+ T cell that is epitope # 3 on the position of amino acids (213-227), # 9A (243-257), # 4 (251-265), # 5 (258-272), # 6 (266-280), # 7 (273-287) which has the same amino acid sequence comparison between strains. At position # 8 epitope (281-295) there are variation of amino acid sequence . Amino acids at positions 500-508 is recognized by CD8 + lymphocytes have the same sequence between strains were compared, and the amino acids at positions 526-531 which recognised by has the same amino acid sequence comparison between strains. Recognition of these epitopes by T lymphocytes and B lymphocytes can be the basis for the development of vaccines, especially vaccines for the Indonesian strain. Cloning of NS3 gene IDS 96/10 were done by using 3 strategies, digestion sticky end of vector and insert, digestion blunt end of vector and digestion blunt end vector then didefosforilation using CIAP. Three of these strategies have not been able to produce a recombinant plasmid pUMVD-4aNS3. Further optimization needs to be done to obtain clones containing the recombinant plasmid.

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ABSTRACTDemam Berdarah Dengue (DBD) adalah penyakit yang disebabkan virus dengan vektor nyamuk yang paling cepat menyebar di duniaGenom DENV terdiri dari tiga protein struktural yaitu capsid (C), protein membran (prM), dan protein envelop (E) serta tujuh gen protein nonstuktural yaitu NS1, NS2a, NS2b, NS3, NS4a,NS4b dan NS5. Protein NS3 mengandung epitop yang dapat dikenali oleh sistem imun humoral maupun selular. Oleh karena itu, protein NS3 merupakan target potensial bagi pengembangan vaksin dengue. Penelitian ini diawali dengan sekuensing pada gen NS3 DENV-4 IDS 96/10. Dari hasil sekuensing dilakukan analisis filogenetik dan analisis epitop. Analisis filogenetik menunjukkan gen NS3 IDS 96 /10 berada dalam satu clade dengan strain yang diisolasi dari Cina (2010), Singapore (2010) dan Thailand (2000). Pada gen NS3 DENV-4 IDS 96/10 terdapat epitop yang dapat dikenali oleh sel limfosit T CD4+ yaitu epitop #3 pada posisi asam amino (213-227) , #9A (243-257), #4 (251-265), #5 (258-272), # 6 (266-280), #7 (273-287) yang mempunyai urutan asam amino sama antar strain yang dibandingkan. Pada posisi epitop #8 (281-295) terdapatvariasi urutan asam amino. Asam amino pada posisi CD8+ mempunyai urutan yang sama antar strain yang dibandingkan,dan asam amino pada posisi 526-531 yang dikenali oleh limfosit B mempunyai urutan asam amino yang sama antar strain yang dibandingkan. Pengenalan epitop- epitop tersebut oleh limfosit T dan limfosit B menjadi dasar pengembangan vaksin khususnya vaksin yang khusus untuk strain Indonesia. Dilakukan pengklonaan gen NS3 IDS 96/10 dengan menggunakan 3 strategi, yaitu dengan digesti vektor dan insert dengan ujung sticky end, digesti vektor dengan ujung blunt end dan digesti vektor dengan ujung blunt end kemudian didefosforilasi menggunakan metode CIAP. Dengan ketiga strategi tersebut belum dapat menghasilkan plasmid rekombinan pUMVD-4aNS3. Perlu dilakukan optimasi lebih lanjut untuk mendapatkan klon yang berisi plasmid rekombinan.Demam Berdarah Dengue (DBD). Penyebab DBD adalahvirus RNA famili flaviviridae yang disebut virus dengue (DENV).500-508 dikenali oleh sellimfosit TCD8+ mempunyai urutan yang sama antar strain yang dibandingkan,dan asam amino pada posisi 526-531 yang dikenali oleh limfosit B mempunyai urutan asam amino yang sama antar strain yang dibandingkan. Pengenalan epitop- epitop tersebut oleh limfosit T dan limfosit B menjadi dasar pengembangan vaksin khususnya vaksin yang khusus untuk strain Indonesia. Dilakukan pengklonaan gen NS3 IDS 96/10 dengan menggunakan 3 strategi, yaitu dengan digesti vektor dan insert dengan ujung sticky end, digesti vektor dengan ujung blunt end dan digesti vektor dengan ujung blunt end kemudian didefosforilasi menggunakan metode CIAP. Dengan ketiga strategi tersebut belum dapat menghasilkan plasmid rekombinan pUMVD-4aNS3. Perlu dilakukan optimasi lebih lanjut untuk mendapatkan klon yang berisi plasmid rekombinan."

"ABSTRAKPendahuluan. VDAC merupakan protein kanal ion yang bertanggung jawab atas aliran ion Ca2+ dan ATP dalam flagela spermatozoa. Defisiensi gen VDAC3 pada mencit dan mutasi gen VDAC3 pada manusia menyebabkan penurunan motilitas spermatozoa, sehingga VDAC3 dapat dijadikan antigen potensial untuk pengembangan vaksin kontrasepsi laki-laki. Tujuan penelitian ini adalah memproduksi protein rekombinan hVDAC3 dari gen hVDAC3 ekson 5-8 spesifik spermatozoa, dan digunakan sebagai antigen untuk produksi antibodi poliklonal pada kelinci.Metode. Gen hVDAC3 ekson 5-8 spermatozoa diperoleh melalui RT PCR, gen disisipkan ke plasmid pET100/D-TOPO dan diklona dalam E coli TOP 10. Analisis gen sisipan dengan PCR, enzim restriksi dan sekuensing DNA. Protein rekombinan hVDAC3 diekspresikan dalam E coli BL21 StarTM (DE3). Karakterisasi protein dilakukan dengan uji Bradford, SDS PAGE, western blot dan purifikasi protein dengan resin Ni-NTA. Antibodi poliklonal diperoleh dengan cara imunisasi protein rekombinan hVDAC3 ke kelinci dan diukur dengan indirect ELISA. Determinasi lokasi hVDAC3 di spermatozoa dengan metode immunoflurosence.Hasil. Amplifikasi PCR gen hVDAC3 ekson 5-8 berukuran 435 pb dan analisis BLAST menunjukkan 100% identik dengan gen VDAC3 manusia dari bank gen. Vektor rekombinan berukuran 6195 pb mengekspresikan protein rekombinan hVDAC3 berukuran 20 kDa. Antibodi poliklonal telah diproduksi kelinci secara bermakna (p<0.05) dengan titer 2.817, dan antibodi dapat berikatan dengan protein hVDAC3 di kepala dan flagela spermatozoa. Selanjutnya, antibodi poliklonal ini akan digunakan dalam pengembangan vaksin kontrasepsi pada laki-laki.

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ABSTRACT

Introduction. Voltage dependent anion channels (VDAC), also known as mitochondrial porins, are group of proteins in mitochondrial outer membrane that allow the passage of metabolites across the mitochondrial outer membrane, and are involved in ions and ATP transport in sperm flagella. Deficiency and mutation of VDAC3 may cause abnormality in structure and motility of human spermatozoa. VDAC3 could be a potential target to develop non hormonal male contraceptive vaccine. The objective of the study was to produce hVDAC3 recombinant proteins from exon 5 to 8 of human sperm VDAC3 spesific gene.

This recombinant protein was subsequenly used as an antigen to produce polyclonal antibodies in rabbits. Methods. hVDAC3 sperm gene obtained by RT PCR, this gene was inserted into plasmid pET 100/D-TOPO and cloned in E coli TOP 10. The gene was analyzed by PCR method, restriction enzymes and DNA sequencing. The proteins expressed in E coli BL21 StarTM (DE3). Characterization of proteins was evaluated by Bradford method, SDS PAGE and western blot. The recombinant protein was purified with NI-NTA resin. Polyclonal antibodies were obtained by immunization of hVDAC3 recombinant protein into rabbits. Indirect ELISA was done to analyze the antibody. Localization of the VDAC3 recombinant protein in human spermatozoa was evaluated by immunofluorescence method.

Result. By doing PCR amplification and BLAST analysis, the study showed that the hVDAC3 gene had 100% identical to hVDAC3 genes in data bank. E coli BL21 StarTM (DE3) containing recombinant vector (6195 bp) expressed the recombinant protein of hVDAC3 in 20 kDa. This protein produced polyclonal antibodies that bound VDAC3 protein on the head and flagella of human spermatozoa."

"Infeksi yang disebabkan oleh virus dengue (DENV) menimbulkan spektrum luas penyakit dari sindrom virus ringan, demam dengue klasik dan penyakit perdarahan berat yaitu demam berdarah dengue (DBD) hingga Dengue Shock Syndrom (DSS). Antibodi terhadap protein struktural E dari serotipe DENV yang heterolog pada infeksi primer dan sekunder tidak dapat menetralkan virus, sehingga walaupun kompleks antibodi-virus difagositosis oleh monosit, DENV tetap dapat bereplikasi di dalam monosit. Kondisi meningkatnya infeksi virus pada sel target diperantarai antibodi ini dikenali sebagai antibody-dependent enhancement (ADE). Protein NS3 merupakan protein terbesar kedua yang dikode oleh genom DENV dan sekuens asam amino primernya merupakan yang paling lestari diantara serotipe DENV yang berguna menghindari ADE. Protein NS3 ditemukan menginduksi respon antibodi dan respon sel T CD4+ dan CD8+, kebanyakan sel T tersebut bereaksi silang antar serotipe. Dilakukan analisis pada epitop sel T dan sel B protein NS3 DENV4 081 yang selanjutnya dilakukan pengklonaan dan ekspresi gen NS3 DENV4 081. Ditemukan posisi epitop sel B 537-544 NS3 DENV4 081 identik dan lestari dengan 124 strain DENV4 di dunia dan dengan keempat serotipe strain Indonesia. Gen NS3 DENV4 081 berhasil diamplifikasi dengan teknik PCR dan berhasil diinsersikan kedalam vektor pQE80L dengan orientasi yang benar. Plasmid rekombinan yang mengandung gen NS3 DENV4 081 ditransformasikan ke dalam E. coli BL21 dan diekspresikan dengan induksi IPTG. Hasil ekspresi protein NS3 DENV4 081 yang ditunjukkan dengan terlihatnya dot yang berwarna lebih pekat pada Dot Blot yang dideteksi dengan detektor anti-His merupakan protein rekombinan NS3 DENV4 081. Hasil Western Blot menunjukkan ekspresi protein rekombinan NS3 yang rendah, sehingga masih perlu penelitian lebih lanjut untuk mengoptimalkan ekspresi protein rekombinan NS3 pada vektor pQE80L.

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Infections caused by dengue virus (DENV) cause a broad spectrum of disease from mild viral syndrome, classic dengue fever to severe hemorrhagic diseases which are dengue hemorrhagic fever (DHF) and Dengue Shock Syndrome (DSS). Antibodies against E protein of heterologous DENV serotypes in primary and secondary infections can not neutralize the virus, although the antibody-virus complexes were phagocytes by monocytes, DENV still replicate inside monocytes. A condition increasing antibody-mediated viral infection on target cell was identified as antibody-dependent enhancement (ADE). NS3 protein is the second largest protein encoded by the genome of DENV and the primary amino acid sequence is the most conserved among DENV serotypes. NS3 protein was found to induce antibody, CD4+ and CD 8+ T cell responses, most of the T cells were cross-reactive between serotypes. Analysis was performed on the T and B cell epitope of NS3 DENV4 081 protein then continue with gene cloning and expression of NS3 DENV4 081. Position of B cell epitope 537-544 NS3 DENV4 081 protein was found identical and conserved to NS3 protein of 124 DENV4 strains around the world and all four serotypes of Indonesia strain. NS3 DENV4 081 gene was successfully amplified by PCR and successfully inserted into the vector pQE80L with the correct orientation. Recombinant plasmid containing NS3 DENV4 081 gene was transformed into E. coli BL21 and expressed by IPTG induction. Results of NS3 DENV4 081 protein expression was indicated by the colored dot produced on Dot Blot detected with anti-His detector. Western Blot result show low NS3 recombinant protein expression, so further research is needed to optimize the NS3 expression in vector pQE80L."

"[ABSTRAKPendahuluan: Infeksi dengue merupakan salah satu penyakit endemik di daerah tropis dan subtropis yang disebabkan oleh virus dengue (DENV). Hingga saat ini belum ada antiviral yang efektif untuk infeksi dengue. Penyebaran dan sirkulasi serotipe DENV berfariasi di setiap lokasi geografi, hal ini menyulitkan dalam melakukan evaluasi vaksin DENV. Oleh karena itu perlu dikembangkan kandidat vaksin DENV menggunakan strain Indonesia supaya dapat memberikan proteksi maksimal. Pada peneltian ini dikembangkan kandidat vaksin DNA tetravalen DENV berbasis gen prM-E DENV strain Indonesia.Metode: Konstruksi plasmid rekombinan kandidat vaksi dilakukan dengan cara menyisipkan gen prM-E setiap serotipe DENV ke dalam vektor pUMVC4a. Gen prM-E DENV merupakan strain Indonesia, yang diamplifikasi dari serum pasien yang terinfeksi dengan virus ini. Kemampuan plasmid rekombinan mengekspresikan protein prM-E DENV diuji di sel mamalia. Kemampuan kandidat vaksin menginduksi respon imun humoral dievaluasi secara monovalen dan tetravalen di mencit jenis ddY. Titer IgG anti dengue diperiksa menggunakan teknik ELISA, sedangkan titer antibodi netralisasi di tentukan dengan uji FRNT. Proteksi vaksin terhadap mencit yang diimunisasi dievaluasi dengan melakukan uji tantang menggunakan sel K562 yang diinfeksi DENV-2. Viremi virus di tentukan dengan menggunakan teknik foccus assay.Hasil: Konstruksi plasmid rekombinan kandidat vaksin DENV-1 dan DENV-3 sudah berhasil dilakukan. Plasmid dapat mengekspresikan protein prM-E DENV di sel mamalia, namun karakteristik dan kinetik protein masih belum dapat diketahui dengan jelas. Keempat kandidat vaksin DNA yang sedang dikembangkan dapat menginduksi respon imun, baik secara monovalen maupun tetravalen. Imunisasi secara tetravalen dapat memberikan proteksi pada mencit yang diuji tantang dengan sel K562 yang diinfeksi dengan DENV-2.;

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ABSTRACTIntroduction: Dengue infections are caused by dengue viruses (DENV) and are endemic in tropical and subtropical regions. At present, there is no effective antiviral treatment for dengue infection. Distribution and circulation of DENV serotypes varies by geographic location, it is difficult to evaluate DENV vaccine. Therefore, it is necessary to develop a vaccine candidate DENV using Indonesian strains in order to provide maximum protection. However, in this study, we constructed a recombinant plasmid-based prM-E gene from the Indonesia strain as a DENV DNA vaccine candidate.Methode: The recombinant plasmid was prepared by inserting the prM-E gene from each DENV serotypes into the plasmid backbone pUMVC4a. prM-E gene an Indonesia strain, which was amplified from patient sera infected with DENV. The ability of the recombinant plasmid expressing the prM-E DENV protein tested in mammalian cells. The ability of candidate vaccines induce humoral immune responses were evaluated monovalent and tetravalent in ddY mice. IgG titers of anti-dengue examined using ELISA technique, while neutralizing antibody titers determined with FRNT test. Vaccine protection against the immunized mice was evaluated by conducting challenge test using K562 cells infected by DENV-2. Viremia was determined by using the foccus assay.Result: Construction of recombinant plasmid vaccine candidate DENV-1 and DENV-3 was successfully performed. Plasmids can express prM-E DENV proteins in mammalian cells, but the characteristics and kinetics of protein still can not clearly known. Fourth DNA vaccine candidate that is being developed to induce an immune response, either monovalent or tetravalent. Tetravalent immunization may provide protection in mice challenged tested with K562 cells infected with DENV-2.;Introduction: Dengue infections are caused by dengue viruses (DENV) and are endemic in tropical and subtropical regions. At present, there is no effective antiviral treatment for dengue infection. Distribution and circulation of DENV serotypes varies by geographic location, it is difficult to evaluate DENV vaccine. Therefore, it is necessary to develop a vaccine candidate DENV using Indonesian strains in order to provide maximum protection. However, in this study, we constructed a recombinant plasmid-based prM-E gene from the Indonesia strain as a DENV DNA vaccine candidate.Methode: The recombinant plasmid was prepared by inserting the prM-E gene from each DENV serotypes into the plasmid backbone pUMVC4a. prM-E gene an Indonesia strain, which was amplified from patient sera infected with DENV. The ability of the recombinant plasmid expressing the prM-E DENV protein tested in mammalian cells. The ability of candidate vaccines induce humoral immune responses were evaluated monovalent and tetravalent in ddY mice. IgG titers of anti-dengue examined using ELISA technique, while neutralizing antibody titers determined with FRNT test. Vaccine protection against the immunized mice was evaluated by conducting challenge test using K562 cells infected by DENV-2. Viremia was determined by using the foccus assay.Result: Construction of recombinant plasmid vaccine candidate DENV-1 and DENV-3 was successfully performed. Plasmids can express prM-E DENV proteins in mammalian cells, but the characteristics and kinetics of protein still can not clearly known. Fourth DNA vaccine candidate that is being developed to induce an immune response, either monovalent or tetravalent. Tetravalent immunization may provide protection in mice challenged tested with K562 cells infected with DENV-2., Introduction: Dengue infections are caused by dengue viruses (DENV) and are endemic in tropical and subtropical regions. At present, there is no effective antiviral treatment for dengue infection. Distribution and circulation of DENV serotypes varies by geographic location, it is difficult to evaluate DENV vaccine. Therefore, it is necessary to develop a vaccine candidate DENV using Indonesian strains in order to provide maximum protection. However, in this study, we constructed a recombinant plasmid-based prM-E gene from the Indonesia strain as a DENV DNA vaccine candidate.Methode: The recombinant plasmid was prepared by inserting the prM-E gene from each DENV serotypes into the plasmid backbone pUMVC4a. prM-E gene an Indonesia strain, which was amplified from patient sera infected with DENV. The ability of the recombinant plasmid expressing the prM-E DENV protein tested in mammalian cells. The ability of candidate vaccines induce humoral immune responses were evaluated monovalent and tetravalent in ddY mice. IgG titers of anti-dengue examined using ELISA technique, while neutralizing antibody titers determined with FRNT test. Vaccine protection against the immunized mice was evaluated by conducting challenge test using K562 cells infected by DENV-2. Viremia was determined by using the foccus assay.Result: Construction of recombinant plasmid vaccine candidate DENV-1 and DENV-3 was successfully performed. Plasmids can express prM-E DENV proteins in mammalian cells, but the characteristics and kinetics of protein still can not clearly known. Fourth DNA vaccine candidate that is being developed to induce an immune response, either monovalent or tetravalent. Tetravalent immunization may provide protection in mice challenged tested with K562 cells infected with DENV-2.]"