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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."

"Peningkatan ekspresi gen Sox2 dan c-Myc telah dilaporkan memiliki korelasi dengan tingkat keparahan kanker payudara. Sox2 dan c-Myc merupakan faktor transkripsi utama yang berperan dalam proses diferensiasi sel punca kanker. Penelitian ini bertujuan mengkonstruksi gen penyandi pluripotensi Sox2 dan c-Myc ke dalam sel inang Escherichia coli DH5α dengan menggunakan plasmid pET101/D-TOPO. Prinsip kloning yang dilakukan adalah dengan mengklon DNA binding domain dari gen Sox2 dan c-Myc. Untuk mendapatkan DNA Sox2 dan c-Myc, dilakukan reverse-transcriptase polymaerase chain reaction (RT-PCR) dengan menggunakan template mRNA dari sel punca kanker payudara serta aplifikasi dengan PCR untuk mendapatkan fragmen DNA dalam jumlah banyak. Forward primer dan reverse primer yang digunakan dirancang dengan menggunakan data dari NCBI GenBank dan UNIPROT serta program Serial Cloner dan PerlPrimer. Sebelum dikloning, dilakukan sekuensing. Hasil sekuensing dianalisis dengan menggunakan BLAST. Fragmen DNA diligasi berdasarkan prinsip penambahan empat basa pada forward primer (CACC) yang overhang terhadap ujung 5' vektor kloning (GTGG). Hasil ligasi ditransformasi menggunakan metode secara kimia dengan CaCl2 dan heat shock. Koloni yang tumbuh direplika dan dilakukan isolasi plasmid. Hasil PCR plasmid rekombinan menunjukkan bahwa gen Sox2 dan c-Myc berhasil disisipkan ke dalam vektor.

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Increase in Sox2 and c-Myc gene expression have been reported to correlate with the severity of breast cancer. Sox2 and c-Myc is the major transcription factor in the process of stem cell differentiation. The objective of this study is to construct the gene coding of pluripotency, Sox2 and c-Myc, into Escherichia coli DH5α host cell by using the pET101/D-TOPO vector. The cloning principle is to clone the binding site domain of Sox2 and c-Myc. In order to get the Sox2 and c-Myc DNA, reverse-transcriptase polymerase chain reaction (RT-PCR) was carried out using mRNA template from breast cancer stem cell and amplification using PCR to obtain DNA fragment in large quantities. Forward primer used were designed using the data from NCBI GenBank and UNIPROT with Serial Cloner and PerlPimer program. Sequencing was carried out before the cloning process. The sequencing result were analyzed using BLAST. DNA frgament was ligated using principle of four base addition to the forward primer (CACC) which overhang in the 5' and of cloning vector (GTGG). The ligation product was transformed using the chemical method with CaCl2 and heat shock. The colonies were replicated and the plasmid was isolated. The result showed that Sox2 and c-Myc was successfully inserted into the vector."

"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."

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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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"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."

"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."

"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."

"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."

"ABSTRAKLatar Belakang : Proses pematangan spermatozoa terjadi melalui interaksi spermatozoa dengan protein yang disekresikan ke lumen oleh sel-sel epitel epididimis. Sekresi protein akan menciptakan lingkungan yang mendukung proses pematangan spermatozoa. Namun gen penyandi protein yang terlibat dalam proses pematangan spermatozoa di epididmis masih belum banyak diketahui. Berdasarkan penelitian sebelumnya, gen-gen yang terlibat dalam proses pematangan spermatozoa ini memiliki kriteria antara lain protein sekretori, terekspresi spesifik di epididimis, dan menunjukkan eskpresi regional, diregulasi oleh faktor androgen dan faktor testikular. Defb30 merupakan salah satu gen yang perlu dilakukan karakterisasi lebih lanjut untuk mengetahui apakah gen tersebut memenuhi kriteria sebagai gen yang terlibat dalam proses pematangan spermatozoa. Tujuan dari penelitian ini adalah untuk melakukan karakterisasi gen Defb30 pada epididimis mencit.Desain : Penelitian ini menggunakan analisis bioinformatika dan Quantitative real-time PCR qRT-PCR .Metode : Analisis bioinformatika digunakan untuk memprediksi struktur gen, sinyal peptida dan domain fungsional. Analisis qRT-PCR digunakan untuk mengukur ekspresi relatif gen Defb30 terhadap analisis sebaran jaringan, regulasi terhadap androgen dan faktor testikular serta postnatal development.Hasil : Analisis sinyal peptida menggunakan signalP 4.1 menunjukkan bahwa Defb30 merupakan protein sekretori. Defb30 terekspresi secara spesifik di epididimis dan memiliki nilai spesifitas tinggi di bagian kaput epididimis. Ekspresi relatif gen Defb30 diregulasi oleh faktor endokrin berupa androgen, penurunan ekspresi relatif gen Defb30 terlihat pada hari pertama hingga hari ketiga gonadektomi dan testosteron diketahui mampu mencegah penurunan ekspresi Defb30 pada mencit yang telah digonadektomi. Analisis eksperimen efferent duct ligation menunjukkan gen Defb30 diregulasi oleh faktor testikular. Analisis postnatal development menunjukkan bahwa gen Defb30 mulai terekspresi pada hari ke-15 postnatal dan meningkat hingga usia dewasa.Kesimpulan : Defb30 merupakan protein sekretori yang terekspresi spesifik pada kaput epididimis dan diregulasi oleh androgen dan faktor testikluar.

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ABSTRACTBackground The process of sperm maturation occurs through interaction between sperm and proteins secreted by epididymal epithelial cells. The secretion of proteins will create micro environment suitable for spermmaturation. However, the role of protein encoding genes involved in the maturation process are not widely known. Based on previous studies the genes that are involved in spermmaturation process have characteristics such as secretory protein, specific expression in the epididymis and shows region specific expression, regulated by androgen and testicular factors. Defb30 is one of the genes that need further characterization to determine the putative function. Therefore, this study was aimed to characterize expression and regulation of Defb30 in the mouse epididymis.Methods Bioinformatics analysis was used to predict the structure of genes, peptide signals and functional domains. qRT PCR analysis was performed to measure the level of Defb30 expressionin the tissue distribution, regulation of andorgen and testicular factors and postnatal development.Result Peptide signal analysis using signalP 4.1 indicated that Defb30 was a secretory protein. Defb30 was expressed exclusively in the epididymis and had a high specificity in the caput. The expression of the Defb30 gene was regulated by androgen in which decreased of Defb30 expression was observed at the first day to the third day of gonadectomy and exogenous T was able to maintain Defb30 expression at 3d and 5d gonadectomized mice. efferent duct ligation showed that Defb30 was slightly regulated by testicular factors. Defb30 was developmentally regulated being expressed start at day 15 postnataly.Conclusions Defb30 is a secretory protein which is expressed specifically in the caput epididymis and it is regulated by androgen and testicular factors. "

"Peran protein retinoblastoma (pRb) dalam pencegahan pembentukan tumor diinhibisi oleh interaksinya dengan protein E7 HPV pada kanker serviks. Oleh sebab itu, perlu dilakukan strategi pengembangan uji in vitro untuk analisis interaksi pRb dan E7, terutama dalam pengembangan vaksin HPV berbasis antigen E7. Protein pRb dapat diperoleh dalam bentuk protein rekombinan yang diproduksi pada bakteri Escherichia coli. Penelitian bertujuan untuk memperoleh klona gen RB1 dalam vektor pQE_80L. Sintesis gen RB1 (2787 pb) dilakukan dengan metode PCR overlap extension. Fragmen gen RB1 dan vektor didigesti dengan enzim restriksi BamHI dan SalI kemudian diligasikan dengan enzim T4 ligase. Hasil ligasi ditransformasi ke dalam Escherichia coli TOP10 secara kejut panas. Hasil transformasi diseleksi menggunakan PCR koloni untuk mengidentifikasi keberadaan DNA sisipan. Sebanyak 1 dari 27 koloni yang diseleksi mengandung plasmid rekombinan. Plasmid rekombinan kemudian diisolasi dan diverifikasi dengan digesti dan sekuensing. Hasil analisis digesti dan sekuensing menunjukkan gen RB1 berhasil disisipkan ke vektor pQE_80L. Namun terdapat beberapa mutasi, yaitu substitusi (c.117G>A dan c.2316T>C) serta mutasi delesi (c.719_724delAAACAG).

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The role of human retinoblastoma protein (pRb) as tumor suppressor is inhibited by its interaction with HPV E7 protein in cervical cancer. Therefore, it is interesting to develop strategy for development of in vitro assay to analyze pRb and E7 interaction, especially in the development of therapeutic HPV vaccine that is based on E7 antigen. The pRb protein can be provided in the form of recombinant protein that is poduced in Escherichia coli. The study objective was to obtain RB1 gene clone in pQE_80L vector. The synthesis of RB1 gene (2787 pb) was performed by using overlap extension PCR. The RB1 gene fragment and vector was digested by BamHI and SalI restriction enzyme then ligated by T4 ligase enzyme. The ligation product was transformed into Escherichia coli TOP10 with heat shock. The transformation result was screened using colony PCR to identify the presence of insert DNA. There was 1 out of 27 selected colonies that carried the recombinant plasmid. The recombinant plasmid then isolated and verified with digestion and sequencing. The results of digestion and sequencing analysis showed that RB1 gene was successfully inserted into pQE_80L vector. However, there were mutations which were substitution (c.117G>A and c.2316T>C) and deletion (c.719_724delAAACAG)."