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"Spag11a diketahui terekspresi secara spesifik pada kaput epididimis sehingga dimungkinkan protein tersebut memiliki fungsi yang spesifik untuk maturasi spermatozoa. Studi peran SPAG11A dalam maturasi spermatozoa di epididimis memerlukan produksi protein SPAG11A untuk dikarakterisasi. Tujuan dari penelitian ini adalah untuk mengklon, mengekspreesikan, dan mengkarakterisasi sifat antimikroba dari protein rekombinan SPAG11A. Insert cDNA Spag11a yang dihasilkan melalui PCR diklon ke dalam vektor pET100/D-TOPO. Plasmid rekombinan kemudian diekspresikan ke Escherichia coli BL21 DE3 star. Deteksi dari fusi protein rekombinan dilakukan dengan SDS-PAGE dan Western Blotting. IMAC Immobilized Metal Affinity Chromatography digunakan untuk mempurifikasi protein rekombinan. Uji antimikroba protein rekombinan dianalisis melalui pengukuran Optical density.PCR amplifikasi dari cDNA kaput epididimis mencit menghasilkan insert Spag11a berukuran 210bp. Insert tersebut kemudian dikloning ke dalam pET100/D-TOPO menghasilkan 1 rekombinan plasmid dari 10 koloni yang diskrining. Ekspresi rekombinan klon ke dalam E.coli BL21 menghasilkan fusi protein setelah diinduksi IPTG selama 4 jam. Fusi protein dikonfirmasi menggunakan Western Blotting menggunakan antibodi yang mengenali N-terminal His-Tag 21kDa dan protein SPAG11A. Uji antimikroba protein rekombinan SPAG11A mununjukkan tidak ada inhibisi yang signifikan terhadap laju pertumbuhan E.coli dan Bacillus subtilis. Insert Spag11a yang berukuran 210bp berhasil diklon ke dalam vektor pET100/D-TOPO. Ekspresi rekombinan Spag11a menghasilkan fusi protein berukuran 21kDa. Protein rekombinan SPAG11A tidak membawa sifat antimikroba terhadap E.coli dan B. subtilis.

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Spag11a is known to be specifically expressed in the caput region of the epididymis suggesting a specific function for sperm maturation. Study of SPAG11A role in the epididymal sperm maturation requires generating SPAG11A protein for characterization. The objective of this study was to clone, express and characterize antimicrobial property of the recombinant SPAG11A. Spag11a cDNA insert was generated by PCR and cloned in TOPO vector. Recombinant DNA plasmid was subsequently expressed in E coli BL 21 star. Detection of recombinant fusion protein was carried out using SDS PAGE and western immunobloting. IMAC Immobilized Metal Affinity Chromatography was used to purify recombinant protein. Optical density measurement was used to analyse antimicrobial property of the recombinant protein. PCR amplification of mouse caput epididymis cDNA produced a 210 bp insert of Spag11a. Cloning of the insert into TOPO pET100 resulted in 2 recombinants out of 10 colonies that were screened. Expression of recombinant clones in the E coli BL21 produced a fusion protein after being induced IPTG for 4 hours. Fusion protein was confirmed by western immunobloting using two antibodies recognizing N terminal His Tag 21 kDa and SPAG11A protein. Antimicrobial assay for SPAG11A recombinant showed no significant inhibition towards growth rates of E coli and Bacillus subtilis. A 210 bp Spag11a insert was successfully cloned into TOPO pET100 vector. Expression of recombinant spag11a produced a fusion protein of 21 kDa. SPAG11A recombinant protein does not have antimicrobial property towards E coli and B subtilis."

"ABSTRAKProses pematangan spermatozoa terjadi karena adanya interaksi antara protein dengan membran plasma spermatozoa. Walaupun proses pematangan spermatozoa ini sangat penting, namun gen yang berperan dalam sekresi protein di epididimis ini masih banyak yang belum dikarakterisasi. Gen-gen yang berperan dalam proses pematangan spermatozoa umumnya merupakan protein sekretorik, terekspresi pada segmen spesifik, diregulasi androgen, faktor testikular dan perkembangan postnatal. Pada penelitian sebelumnya diketahui bahwa b-defensin merupakan gen yang banyak terekspresi di organ reproduksi pria dan memiliki peran dalam pertahanan tubuh dan pematangan spermatozoa. Penelitian ini dilakukan untuk mengkarakterisasi ekspresi gen Defb20 untuk mengetahui perannya dalam proses pematangan spermatozoa. Studi in silico dilakukan untuk prediksi struktur gen, signal peptide dan domain fungsional. Quantitative real-time PCR digunakan untuk mengukur ekspresi gen Defb20 pada analisis sebaran jaringan, regulasi androgen dan faktor testikular serta postnatal developmen. Hasil penelitian mendapatkan bahwa sekuen Defb20 mengandung domain penting seperti N-myristoilation dan beberapa situs phosporilasi protein kinase yang mungkin berperan dalam mekanisme interaksi protein dengan membran plasma. Sekuen asam amino Defb20 mengandung signal peptides, mengindikasikan protein yang disekresikan dan terlibat dalam proses pematangan spermatozoa. -defensins 20 (Defb20) terekspresi spesifik di epididimis dengan ekspresi tertinggi terdapat pada kaput epididimis. Defb20 diregulasi oleh androgen yang ditunjukkan dengan adanya penurunan ekspresi Defb20 paska dilakukan gonadektomi dan kondisi ini dapat diperbaiki dengan pemberian hormon pengganti. Defb20 juga diregulasi oleh faktor testikular, yang dibuktikan dari menurunnya ekspresi Defb20 setelah ligasi pada duktus eferen (efferent duct ligation (EDL)). Defb20 mulai terekspresi pada hari ke-21 setelah lahir yang mengindikasikan gen Defb20 terekspresipada suatu periode perkembangan epididimis. Berdasarkan hasil penelitian disimpulkan bahwa Defb20 memiliki karakteristik ekspresi : mengandung signal peptide yang mengarahkan sintesis protein pada jalur sekretorik, spesifik terekspresi di epididimis, diregulasi androgen dan faktor testikular serta mulai terekspresi pada masa pubertas hingga dewasa

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ABSTRACTEpididymal sperm maturation occurs via interactions between sperm and proteins secreted by the epididymal epithelium. Although this is an important process, the genes that encode secreted proteins remain largely uncharacterized. The genes that play a role in sperm maturation process has character, among others; is a secretory protein, expressed specifically in the epididymis, regulated by androgen, testicular factor, and postnatal development. Previous studies showed that family of-defensins preferentially eaxpressed in male reproductive tracts and play an important role in both innate immunity and sperm fertility. This study aimed to characterize Defb20 to understand its role in sperm maturation. This study using in silico analyses and quantitative real-time PCR (qRT-PCR). In silico analyses were performed to predict gene structure, signal peptides and functional domains. Defb20 expression in various tissues, after gonadectomy, efferent duct ligation and postnatal development were measured using quantitative real-time RT-PCR. Defb20 sequence contains important domains such as N-myristoilation and kinase binding sites which are putatively involved in the protein activation and protein-plasma membrane interaction. The amino acid sequence of Defb20 contains signal peptides, indicating characteristic of secretory proteins involved in the sperm maturation. β-defensins 20 (Defb20) was expressed exclusively in the epididymis with the highest expression in the caput region. Defb20 was regulated by androgen showing down-regulation after gonadectomy and the expression was recovered after testosterone replacement. However, Defb20 was also regulated by testicular factors in which the expression was down-regulated after efferent duct ligation (EDL). The dependency on the androgen was further confirmed by postnatal expression analysis in which Defb20 begin to express at day21 postnatal indicating specific stage of expression after initial development of the epididymis. In conclusion, Defb20 have a potential to be involved in the epididymal sperm maturation process. Defb20 has characteristic expression; has a signal peptide sequence that directs synthesis in the secretory pathway, specifically expressed in the epididymis, androgen and testicular factors regulated, and expressed in puberty to adulthood"

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Latar Belakang: Beberapa gen yang terekspresi spesfik di epididimis diduga

terlibat dalam proses pematangan sperma. Karakteristik gen yang terlibat dalampematangan sperma selain ekspresinya spesifik di epididimis juga dipengaruhioleh androgen, faktor testikuler, dan terekspresi pada saat masa pubertas. Salahsatu famili gen yang cukup banyak ditemukan terekspresi di epididimis adalahBeta Defensin. Gen Beta Defensin diketahui memiliki peran sebagai pertahananterhadap mikroba, namun diduga memiliki keterlibatan dalam proses pematangansperma karena ekspresinya banyak ditemukan di epididimis. Oleh karena itu,penelitian pada gen Beta Defensin terhadap perannya dalam proses pematangansperma perlu dilakukan. Berdasarkan studi sebelumnya diketahui bahwa salahsatu gen Beta Defensin yang terekspresi di epididimis yaitu Beta Defensin 2(Defb2), namun karakterisasi terhadap gen ini belum dilakukan. Dengandemikian, pada penelitian ini bertujuan untuk mengkarakterisasi gen Defb2 terkaitdengan perannya pada proses pematangan sperma.Metode: Analisis bioinformatika digunakan untuk mendapatkan informasi

mengenai struktur gen, signal peptide, dan domain fungsional pada gen Defb2.

Analisis qRT-PCR untuk mengetahui ekspresi relatif gen Defb2 pada berbagai

jaringan, regulasinya oleh androgen, pengaruh dari faktor testikular dan

ekspresinya pada perkembangan postnatal.Hasil: Defb2 merupakan protein sekretori karena memiliki signal peptide. Defb2

memiliki domain fungsional berupa N-myristoylation dan protein kinase-C. Gen

Defb2 terekspresi spesifik di epididimis khususnya pada bagian caput epididimis.

Defb2 ekspresinya dipengaruhi oleh androgen terbukti setelah perlakuan

gonadektomi, ekspresi Defb2 menjadi menurun dan kembali mengalami kenaikan

ketika diberikan testosteron eksogen. Defb2 juga ekspresinya dipengaruhi oleh

faktor testikuler terbukti setelah diberi perlakuan Efferent Duct Ligation (EDL)

maka ekspresi Defb2 langsung menurun bahkan terjadi apoptosis sel sehingga

pola ekspresi gen Defb2 sudah tidak bisa diamati. Begitu juga pada analisis

postnatal development terlihat ekspresi gen Defb2 mulai terdeteksi jelas pada hari

ke-15 yang merupakan masa pubertas mencit jantan.Kesimpulan: Defb2 merupakan gen yang terlibat dalam proses pematangan

sperma di epididimis yang dibuktikan dengan ekspresi spesifik di epididimis,

diregulasi oleh androgen dan faktor testikuler, serta mulai terekspresi pada masa

pubertas.

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Background: Some of the specific genes expression in the epididymis are

suspected to be involved in the process of sperm maturation. Characteristics of thegenes involved in sperm maturation in the epididymis-specific expression inaddition also influenced by androgens, testicular factors, and expressed at the timeof puberty. One of a family of genes that is pretty much found expressed in theepididymis is a Beta Defensins. Beta Defensin genes known to have a role as adefence against microbes, but suspected to have involvement in the process ofsperm maturation because the expression is found in the epididymis. Therefore,research on Beta Defensin genes against its role in sperm maturation processneeds to be done. Based on previous studies it is known that one of the BetaDefensin genes which expressed in the epididymis that is Beta Defensins 2(Defb2), but the characterization of this gene has not been made against. Thus,this research aims to characterize genes associated with the Defb2 role in theprocess of sperm maturation.Methods: Bioinformatics analysis was used to obtain information about the

structure of genes, signal peptides, and functional domains of the Defb2 gene.

qRT-PCR analysis to find out the relative gene expression of Defb2 on various

tissue, regulation by androgens, the effect of testicular factors and its expression

in postnatal development.Results: Defb2 is a secreted protein because it has signal peptides. Defb2 has a

functional domain in the form of N-myristoylation and kinase-C protein. Specific

genes expression of Defb2 in the epididymis is especially in the caput epididymis.

Defb2 expression influenced by androgens is proven after the gonadectomy, the

expression of Defb2 to be decreased and start increase again when exogenous

testosterone is given. Defb2 also its expression influenced by testicular factors

that proven after being given the treatment by Efferent Duct Ligation (EDL), then

the Defb2 expressions directly decreased and the cell apoptosis occurs even so

that the pattern of gene expression Defb2 already could not be observed. So also

on analysis of postnatal development seen gene expression Defb2 begins to be

detected clearly at day 15 which is a male mice puberty.Conclusions: Defb2 is a gene which is involved in the process of maturation of

sperm in the epididymis that is evidenced by specific expression in the

epididymis, be regulated by androgens and testicular factors, and as well as start

expressed at puberty.

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"Latar belakang: Beta defensin diekspresikan terutama oleh sel epitel pada permukaan mukosa berbagai organ seperti kulit, usus, mulut dan saluran genital. Studi sebelumnya menunjukkan bahwa beta defensin 30 (Defb30) terekspresi spesifik di epididimis. Defb30 merupakan peptida kationik berukuran kecil yang diduga berperan penting pada proses pematangan spermatozoa di epididimis dan juga berperan sebagai pertahanan host terhadap infeksi mikroba. Untuk mempelajari aktivitas antimikroba Defb30 ini diperlukan analisis pada tingkat protein dan hal tersebut memerlukan protein dalam jumlah yang cukup. Karena itu perlu dilakukan suatu rekayasa genetika untuk pembuatan protein rekombinan DEFB30.Metode: Gen sintetik penyandi protein DEFB30 yang telah dioptimasi kodonnya diklona ke dalam vektor pQE-80L, suatu plasmid yang mengandung sistem ekspresi untuk prokariota. Plasmid rekombinan yang mengandung sisipan gen target dikonfirmasi dengan analisis enzim restriksi dan sekuensing. Selanjutnya plasmid rekombinan di ekpresikan ke dalam E. coli BL21 dan diinduksi menggunakan IPTG (Isopropyl-1-Thio-d-Galactopyranoside) dengan berbagai waktu inkubasi. Deteksi protein rekombinan dilakukan dengan SDS-PAGE dan westernblotting. IMAC (Immobilized Metal Affinity Chromatography) digunakan untuk mempurifikasi protein rekombinan. Uji antimikroba protein rekombinan dilakukan dengan cara pengukuran nilai optical density (OD) dan dianalisis hasilnya menggunakan uji one way anova.Hasil: Gen sintetik penyandi protein rekombinan DEFB30 berhasil dikonstruksi pada plasmid pQE-80L. Ekspresi ke dalam E. coli BL21 menghasilkan suatu protein fusi setelah diinduksi menggunakan IPTG selama 4 jam. Hasil analisis protein rekombinan dengan westernblotting menggunakan antibodi Anti-His G-HRP menunjukkan terbentuk pita tebal yang berukuran diatas 10 kDa (±12 kDa). Uji antimikroba protein rekombinan DEFB30 menunjukkan bahwa DEFB30 dapat menghambat pertumbuhan bakteri Eschericia coli dan Bacillus subtilis.Kesimpulan: Gen sintetik penyandi beta defensin 30 berhasil diklona ke dalam plasmid pQE-80L. Ekspresikan protein rekombinan DEFB30 menghasilkan suatu protein fusi berukuran ±12kDa. Protein rekombinan DEFB30 terbukti memiliki sifat antimikroba terhadap Eschericia coli dan Bacillus subtilis.

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ackground: Beta defensins are primarily expressed by epithelial cells at mucosal surfaces, such as those in skin, gut, mouth and genital tracts. Previous studies have demonstrated that beta defensin 30 (Defb30) is exclusively expressed in the epididymis. Defb30 is known as a small cationic antimicrobial peptide which plays an important role in epididymal sperm maturation and also acts as a host defence against microbial infection. Study of Defb30 role in the antimicrobial activity requires generating DEFB30 protein for characterization. For the purpose of this study, genetic engineering was done for the manufacture of the DEFB30 recombinant protein.

Methods: In this study, according to the preferred codon in E. coli, the Defb30 gene was optimized and synthesized. The gene was cloned into pQE-80L vector and subsequently expressed in E. coli BL21; using IPTG (Isopropyl-1-Thio-d-Galactopyranoside) as an inducer. Detection of recombinant protein was carried out by using SDS-PAGE and westernblotting. IMAC (Immobilized Metal Affinity Chromatography) was used to purify recombinant protein. Optical density measurement was used to analyze antimicrobial property of the DEFB30 recombinant protein.

Results: The synthetic gene was successfully constructed into pQE-80L plasmid and expression of the recombinant protein in E. coli BL21 produced a fusion protein after being induced by IPTG for 4 hours. Westernblotting analysis using Anti-His G-HRP antibody showed band above 10kDa (±12kDa). Antimicrobial assay for DEFB30 recombinant protein showed inhibition towards growth rates of Eschericia coli and Bacillus subtilis.

Conclusion: Defb30 synthetic gene was succesfully cloned into pQE-80L plasmid. Expression of recombinant DEFB30 produced a fusion protein of ±12kDa. This recombinant protein has antimicrobial property towards Eschericia coli and Bacillus subtilis."

"ABSTRAKSalah satu kendala yang dihadapi manusia saat ini adalah pertambahan penduduk yang tidak terkendali yang menimbulkan banyak masalah baru di berbagai aspek kehidupan. Oleh sebab itu, pengendalian pertumbuhan penduduk harus dilakukan dengan berbagai metode kontrasepsi. Pengembangan kontrasepsi pria non-hormonal dengan menghambat proses pematangan sperma di epididimis menjadi hal yang menjanjikan. Sayangnya, gen yang berperan di dalam proses pematangan sperma di epididimis masih belum banyak dipelajari. Kami menganalisis beberapa kandidat gen yang diekspresikan di epididimis, salah satunya adalah Defensin Beta-42 (Defb42). Beberapa langkah metode yang kami lakukan adalah isolasi epididimis dan ekstraksi RNA, analisis bioinformatika, dan real-time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) untuk menganalisa ketergantungan ekspresi terhadap androgen dari gen Defb42 karena pematangan sperma bergantung pada androgen. Selain itu, proses pematangan sperma juga terjadi akibat interaksi antara sperma dan protein yang disekresikan oleh epitel epididimis. Oleh sebab itu, peptida sinyal harus dianalisis juga untuk mengecek apabila gen ini adalah protein sekretori. Hasilnya adalah gen ini diregulasi oleh androgen dan memiliki peptida sinyal. Hal ini membuat gen Defb42 menjadi kandidat gen yang menjanjikan untuk diteliti lebih lanjut dalam upaya pengembangan kontrasepsi non-hormonal pada pria.

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ABSTRACTOne of the problem nowadays is the uncontrolled population growth. This raises many other problems in every aspect of life. Therefore, the population growth must be controlled with many types of contraceptive agents. The development of male non-hormonal contraceptive agent by inhibiting the sperm maturation process in epididymis seems to be promising. We analyzed several candidates of gene which are expressed in epididymis; one of them is Defensin Beta-42 (Defb42) gene. Several method we conducted in this research are epididymis isolation and RNA extraction, bioinformatics analysis, and real time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) to analyze the expression dependency towards androgen of Defb42 gene because the sperm maturation is androgen-dependent process. Besides, the sperm maturation process occurs due to the interaction between sperm and protein secreted by epididymal epithelium. Therefore, the signal peptide has to be analyzed to confirm whether the candidate gene is a secretory protein. The results are this gene is regulated by androgen and has signal peptide properties. Therefore we can conclude that the gene is promising to be studied further in effort to develop male non-hormonal contraceptive agent."

"Latar belakang: Beta defensin diekspresikan terutama oleh sel epitel pada permukaan mukosa berbagai organ seperti kulit, usus, mulut dan saluran genital. Studi sebelumnya menunjukkan bahwa Beta defensin 30 (Defb30) terekspresi spesifik di epididimis. Defb30 merupakan peptida kationik berukuran kecil yang diduga berperan penting pada proses pematangan spermatozoa di epididimis dan juga memiliki kemampuan untuk membunuh mikroba. Untuk mempelajari aktivitas antimikroba Defb30 ini diperlukan analisis pada tingkat protein dan hal tersebut memerlukan protein dalam jumlah yang cukup. Karena itu perlu dilakukan suatu rekayasa genetika berupa perancangan gen yang mengkode Defb30, pengklonaan dan ekspresi untuk pembuatan protein rekombinan DEFB30. Metode: Gen sintetik penyandi protein DEFB30 yang telah dioptimasi kodonnya diklona ke dalam vektor pQE-80L. Plasmid rekombinan yang mengandung sisipan gen target dikonfirmasi dengan analisis enzim restriksi dan sekuensing untuk selanjutnya diekpresikan ke dalam E. coli BL21 dan diinduksi menggunakan IPTG (Isopropyl-1-Thio-d-Galactopyranoside) dengan berbagai waktu inkubasi. Deteksi protein rekombinan dilakukan dengan SDS-PAGE dan westernblotting. IMAC (Immobilized Metal Affinity Chromatography) digunakan untuk mempurifikasi protein rekombinan. Uji antimikroba protein rekombinan dilakukan dengan cara pengukuran nilai optical density (OD) dan dianalisis hasilnya menggunakan uji one way anova. Hasil: Gen sintetik penyandi protein rekombinan DEFB30 berhasil dikonstruksi pada plasmid pQE-80L. Ekspresi ke dalam E. coli BL21 menghasilkan suatu protein fusi setelah diinduksi menggunakan IPTG selama 4 jam. Hasil analisis protein rekombinan dengan westernblotting menggunakan antibodi Anti-His G-HRP menunjukkan terbentuk pita tebal yang berukuran diatas 10 kDa (±12 kDa). Uji antimikroba protein rekombinan DEFB30 menunjukkan bahwa protein tersebut dapat menghambat pertumbuhan bakteri Eschericia coli dan Bacillus subtilis.Kesimpulan: Gen sintetik penyandi beta defensin 30 berhasil diklona ke dalam plasmid pQE-80L. Ekspresi protein rekombinan DEFB30 menghasilkan suatu protein fusi berukuran ±12kDa. Protein rekombinan DEFB30 terbukti memiliki sifat antimikroba terhadap Eschericia coli dan Bacillus subtilis.

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Background: Beta defensins are primarily expressed by epithelial cells at mucosal surfaces, such as those in skin, gut, mouth and genital tract. Previous studies have demonstrated that beta defensin 30 (Defb30) is exclusively expressed in the epididymis. Defb30 is known as a small cationic antimicrobial peptide which plays an important role in epididymal sperm maturation and also acts as a host defence against microbial infection. Study of Defb30 role in the antimicrobial activity requires generating DEFB30 protein for characterization. For the purpose of this study, Defb30 gene was designed, synthesized, cloned, and expressed for the manufacture of the DEFB30 recombinant protein. Method(s): In this study, according to the preferred codon in E. coli, the Defb30 gene was optimized and synthesized. The gene was cloned into pQE-80L vector and subsequently expressed in E. coli BL21; using IPTG (Isopropyl-1-Thio-d-Galactopyranoside) as an inducer. Detection of recombinant protein was carried out by using SDS-PAGE and westernblotting. IMAC (Immobilized Metal Affinity Chromatography) was used to purify recombinant protein. Optical density measurement was used to analyze antimicrobial property of the DEFB30 recombinant protein. Results: The synthetic gene was successfully constructed into pQE-80L plasmid and expression of the recombinant protein in E. coli BL21 produced a fusion protein after being induced by IPTG for 4 hours. Westernblotting analysis using Anti-His G-HRP antibody showed band above 10kDa (±12kDa). Antimicrobial assay for DEFB30 recombinant protein showed inhibition towards growth rates of Eschericia coli and Bacillus subtilis. Conclusion: Defb30 synthetic gene was succesfully cloned into pQE-80L plasmid. Expression of recombinant DEFB30 produced a fusion protein of ±12kDa. This recombinant protein has antimicrobial property towards Eschericia coli and Bacillus subtilis."

"Background: VP6 protein is an intermediate layer on rotavirus outer capsid shell which is the major structural protein and play an important role in replication cycle. VP6 protein is a conserved region that can induce immune response as target protein of T cell with cross-reactive epitopes within another genotypes.Objectives: This research conducted to determine the molecular characterization of rotavirus VP6 recombinant protein of Indonesia strain, and to determine the clone and expression of VP6 protein in Escherichia coli BL21 for development of rotavirus vaccine.Methods: Rotavirus RNA was extracted from clinical sample of R55 and R10 strains that having correlation with genotipes I and II rotavirus. RNA samples were amplified with RT-PCR reaction and produced 1194 bp amplicon, and sequencing reaction were conducted to confirm and analyze the molecular characterization of VP6 protein in bioinformatics. VP6 gene as insert and pQE-80L plasmids as vector were double restricted and then ligated by ligation enzyme. Product of ligation were transformed to E.coli Top 10 competent cells and the clones were selected to get the recombinant plasmids which bearing VP6 gene. The recombinant plasmid than subcloned to E.coli BL21 competent cells and induced by IPTG and its pellets were loaded directly onto SDS-PAGE, approximately 45 kDa protein was observed on the SDS-PAGE. The protein was analyzed using Western-blotting.Results: Level of amino acids homology from R55 and R10 rotavirus strain compared with vaccine strains and vaccine candidate strains showed high level of homology, with the conserved regions of T-cell epitopes. R55 strain having close relativity with genotype I while R10 strain having close relativitywith genotype II. there are the same level of hydrophobicity between R55 and R10 which indicated as surface proteins or as a hydrophilic protein. The differences of secondary structure between R55 and R10 in amino acids position of 149-152 and 341-349. The VP6 cloned obtained from E.coli Top 10 with pQE-80L plasmid. The profile of expressed VP6 recombinant protein from R55 and R10 strains in SDS-PAGEshowed the different intensity of protein between induced condition with IPTG and non-induced condition, indicated that VP6 protein might be successfully expressed.The Western-Blot assay showed the same result between the cell that induced and non-induced, but it still need another confirmation.Conclusion: The resultof molecular characterization from VP6 recombinant protein and the cloned of VP6 gene that obtained from R55 and R10 rotavirus strains of Indonesia could beapplied as a preliminary study to develop rotavirus candidate vaccine based on subunit vaccine.

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Latar belakang: Protein VP6 rotavirus adalah protrein struktural utama yang berperan penting selama replikasi, danmerupakan bagian yang paling lestaridan memiliki potensi dalam menstimulasi respon imun, yaitu sebagai protein target yang dapat menstimulasi sel T, dan memiliki epitop yang cross-reactive di antara genotipe rotavirus lainnya, sehingga memiliki potensi untuk dikembangkan sebagai vaksin.Tujuan: Untuk mengetahui karakterisasi molekular protein VP6 rotavirus strain Indonesia, serta pengklonaan gen dan ekspresi protein VP6 pada Escherichia coli BL21 untuk pengembangan vaksin rotavirus.Metode: RNA rotavirus R55 dan R10 diperoleh dari ekstraksi sampel klinis. RNA tersebut kemudian diamplifikasi dengan reaksi RT-PCR dan menghasilkan amplikon 1194 bp yang selanjutnya disekuensing untuk konfirmasi dan mengetahui karakterisasi molekular protein VP6 secara bioinformatika. Gen VP6 sebagai sisipan dan plasmid pQE-80L sebagai vektor direstriksi ganda dengan enzim restriksi dan diligasi menggunakan enzim ligasi. Produk ligasi ditransformasikan pada sel kompeten E.coli Top 10 dan diseleksi klon pembawa plasmid rekombinan. Plasmid rekombinan yang mengandung gen VP6 ditransformasikan ke sel kompeten E.coli BL21. Ekspresi protein dilakukan dengan induksi IPTG. Hasil ekspresi dianalisis dengan SDS-PAGE dan dikonfirmasi dengan Uji Western Blot.Hasil: Sekuen asam amino gen VP6 rotavirus strain Indonesia R55 dan R10 memiliki tingkat homologi yang tinggi dengan epitopyang lestari bila dibandingkan dengan strain vaksin dan kandidat vaksin rotavirus; strain R55 lebih dekat kekerabatannya dengan rotavirus genotipe I, strain R10 lebih dekat kekerabatannya dengan rotavirus genotipe II. Sekuen VP6 rotavirus strain R55 dan R10 menunjukkan tingkat hidrofobisitas yang sama, hal ini mengindikasikan sejenis protein permukaan atau protein yang bersifat hidrofilik. Hasil analisa struktur sekunder pada strain R55 dan R10 menunjukkan adanya perbedaan pada posisi asam amino 149-152 dan 341-349. Telah didapatkan klon pQE-80L yang mengandung gen VP6 dari strain R55 dan R10. Ekspresi protein VP6 pada SDS-PAGE menunjukkan adanya perbedaan intensitas pita protein antara sel E. coli yang diinduksi IPTG dengan yang tidak diinduksi, mengindikasikan protein VP6 diduga berhasil diekspresikan. Konfirmasi ekspresi protein menggunakan Western-Blot menunjukkan hasil yang sama antara sel yang diinduksi dengan yang tidak diinduksi, namun hasil ini perlu dikonfirmasi lebih lanjut.Kesimpulan: Hasil karakterisasi molekular protein VP6 rekombinan dan pengklonaan gen VP6 dari rotavirus strain Indonesia R55 dan R10 dapat dikembangkan sebagai studi awal pada pengembangan vaksin subunit berbasis protein VP6 rekombinan. Namun untuk tahap ekspresi protein rekombinan VP6 perlu optimasi lebih lanjut."

"ABSTRAKLatar Belakang: Pematangan spermatozoa di epididimis terjadi melalui interaksi antara spermatozoa dengan protein yang disekresikan oleh sel epitel yang melapisi duktus epididimis. Sekresi protein tersebut menciptakan microenvironment yang diregulasi oleh gen-gen tertentu. Studi sebelumnya menunjukkan bahwa gen yang terlibat dalam pematangan spermatozoa pada umumnya terekspresi secara spesifik di epididimis dan dipengaruhi oleh androgen. Spink2 merupakan salah satu gen yang terekspresi di epididimis, namun regulasi ekspresinya masih belum diketahui. Tujuan penelitian ini adalah untuk mengkarakterisasi ekspresi dan regulasi gen Spink2 pada epididimis mencit jantan.Metode: Analisis secara in silico digunakan untuk mengetahui struktur gen dan prediksi sinyal peptida, serta domain fungsional gen Spink2. Quantitative real-time RT-PCR digunakan dalam mengukur ekspresi relatif gen Spink2 pada analisis spesifisitas jaringan, ketergantungan terhadap androgen dan faktor testikuler, serta post-natal development.Hasil: Spink2 termasuk dalam famili serine protease inhibitor yang ditandai dengan adanya domain Kazal type 2. Analisis signal peptide menunjukkan bahwa Spink2 merupakan protein sekretori. Spink2 terekspresi di testis dan epididimis, dengan ekspresi tertinggi berada di kaput epididimis. Ekspresi Spink2 pada mencit yang digonadektomi mengalami peningkatan setelah 6 jam, kemudian menurun mulai dari hari ke-1 hingga hari ke-5. Pemberian testosteron mampu mempertahankan ekspresi Spink2 pada 3 dan 5 hari setelah gonadektomi. Selain itu, pada analisis pengaruh faktor testikuler, ekspresi Spink2 menunjukkan adanya regulasi dari faktor testikuler pada semua kelompok setelah dilakukan efferent duct ligation EDL . Spink2 menujukkan regulasi post-natal yakni mulai terekspresi pada umur mendekati 22 hari.Kesimpulan: SPINK2 merupakan protein sekretori yang terekspresi pada kaput epididimis, serta diregulasi oleh androgen dan faktor testikuler. Spink2 tidak terekspresi secara konstitutif. Berdasarkan data tersebut Spink2 sangat berpotensi terlibat dalam proses pematangan spermatozoa di epididimis. Penelitian lebih lanjut diperlukan untuk mengkonfirmasi potensi tersebut.

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ABSTRACTBackground Sperm maturation in the epididymis occurs through interactions between sperm and proteins secreted by epithelium cells lining the epididymal duct. The secretion of these proteins creates a microenvironment that is regulated by certain genes. Previous studies showed that genes which are involved in sperm maturation process are expressed specifically in the epididymis and regulated by androgen. Spink2 is one of the epididymal genes, but the regulation of its expression is still unknown. Therefore, this study was aimed to characterize Spink2 expression and its regulation in the mouse epididymis.Method s In silico analysis was performed to determine the gene structure and identify the signal peptide, as well as the functional domain of Spink2. Quantitative real time RT PCR was performed to measure relative expression of Spink2 in the analyses of the tissue specificity, androgen dependency, testicular factor and post natal development.Result s Spink2 belongs to the serine protease inhibitor family which is characterized by the presence of Kazal type 2 domain. Signal peptide analysis showed that Spink2 amino acid sequence contains a signal peptide, indicating Spink2 is a secretory protein. Spink2 was expressed specifically in the testis and epididymis, with the highest level of its expression was in the epididymal caput. Spink2 expression increased after six hours and started to decrease on day 1 throughout day 5. Interestingly, administration of exogenous testosterone was able to maintain expression at the physiological level. In addition, Spink2 was slightly affected by testicular factors. During post natal development, Spink2 start to be expressed at day 22 before increased dramatically throughout day 60.Conclusion s Spink2 is a secretory protein that is expressed in caput region of the mouse epididymis and regulated by androgen. Spink2 is not constitutively expressed throughout development. Based on our data, may be involved in epididiymal sperm maturation process. Further studies are required to confirm its role. "

"Potensi keanekaragaman Indonesia memberikan peluang untuk mendapatkan mikroorganisme penghasil endo-β-1,4-glukanase yang mampu menghidrolisis selulosa. Bacillus amyloliquefaciens BPPTCC-RK2 telah berhasil diisolasi dari rayap. Gen endo-β-1,4-glukanase dikloning dari DNA genom B.amyloliquefaciens BPPTCC-RK2 menggunakan metode rekombinatorial dan diekspresikan secara fungsional di dalam E.coli. Didapatkan ORF sepanjang 1500 nukleotida yang menyandikan 499 asam amino dengan berat molekul 55 kDa. Gen dikloning kedalam pDEST14 dan dioverekspresi pada E.coli BL21-Star. Aktivitas tertinggi sebesar 26,05 U/mg protein setelah diinduksi dengan 1mM IPTG selama 24 jam. Enzim optimum pada pH 6,0 dan suhu 65℃ dan memiliki waktu paruh 90 menit pada suhu 60℃. Pada konsentrasi etanol 100 g/L, masih memberikan aktivitas hingga 78% setelah 24 jam.

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Indonesia has potential biodiversity that provides opportunities to obtain endo-β-1,4-glucanase producing microorganism that could hydrolize cellulose. Bacillus amyloliquefaciens BPPTCC-RK2 have been isolated from termites. Endo-β-1,4-glucanase gene have been cloned from genomic DNA B.amyloliquefaciens BPPTCC-RK2 using recombinatorial method and functionally expressed in E.coli. A full length gene of endo-β-1,4-glucanase consisting 1500 nucleotides that encoded for a protein 499 amino acids with predicted molecular weight 55 kDa. Highest enzyme activity (26,05 U/mg) achieved after 24 hour induction with 1mM IPTG. The enzyme optimum at pH 6,0 and temperature 65℃ and 90 minutes half-life at 60℃. This enzyme give 78% residual activity after 24 hour incubation in 100 g/L ethanol."

"Bakteri Weissella confusa MBF 8-1 yang diisolasi dari produk ampas kacang kedelai terfermentasi telah diteliti memiliki aktivitas Bacteriocin Like Inhibitory Substance (BLIS) terhadap bakteri Leuconostoc mesenteroides. W. confusa MBF8-1 menyandikan tiga jenis bakteriosin yaitu bakteriosin 1 (Bac1), 2 (Bac2), dan 3 (Bac3). Di masa depan, diharapkan bakteriosin tersebut dapat digunakan sebagai peptida antimikroba baru maupun sebagai komplemen antibiotik. Penelitian ini bertujuan untuk menghasilkan vektor rekombinan pembawa gen bakteriosin 1 (bac1) yang dapat diintroduksi ke inang yang sesuai. Vektor rekombinan dikloning dengan metode rekombinatorial Gateway®. Amplifikasi bac1 dengan teknik PCR menggunakan primer yang didesain spesifik dari sekuens bac1 dengan tag attB. Produk PCR disisipkan ke plasmid pDONRTM221 lewat reaksi BP. Plasmid rekombinan selanjutnya ditransformasikan ke sel inang Escherichia coli DH5α. Keberadaan bac1 pada plasmid rekombinan diverifikasi dengan sekuensing. Transformasi yang dilakukan berhasil mengkloning bac1 ke vektor rekombinan, sehingga diperoleh plasmid pENT_Wcbac1 yang dapat digunakan untuk proses selanjutnya dalam ekspresi Bac1.

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Weissella confusa MBF 8-1 was isolated from waste of fermented soya and showed Bacteriocin Like Inhibitory Substance (BLIS) activity against bacteria Leuconostoc mesenteroides. There are three types of bacteriocin produced by W. confusa MBF8-1: bacteriocin 1 (Bac1), 2 (Bac2), and 3 (Bac3). In the future, bacteriocin is potent either to be a new antimicrobial peptide or as antibiotics complement. This experiment was conducted to clone recombinant vector containing bacteriocin 1 gene (bac1) that later can be introduced to suitable expression system. Recombinant vector was cloned by Gateway® recombinatorial technique. First, bac1 was amplified by PCR, using specifically designed primers from bac1 sequence added with attB tag. The PCR product then inserted into pDONRTM221 by BP recombination reaction. Finally, the resulting recombinant plasmid was transformed to Escherichia coli DH5α. The bac1 was verified by sequencing. The transformation successfully cloned bac1 into recombinant vector, named pENT_Wcbac1, which later can be used in the next step of Bac1 expression."