Path: TopS1-Final ProjectPharmacy2008

KLONING KERANGKA BACA TERBUKA GEN PENGKODE VP28 WHITE SPOT SYNDROME VIRUS PADA Escherichia coli DH5a

CLONING OF VP28 OPEN READING FRAME FROM WHITE SPOT SYNDROME VIRUS IN Escherichia coli DH5a

Undergraduate Theses from JBPTITBPP / 2009-04-15 18:42:26
Oleh : FAUSTINE MARCELINE (NIM 10704035), S1 - Department of Pharmacy
Dibuat : 2008, dengan 8 file

Keyword : white spot syndrome virus (WSSV), budidaya udang, protein VP28, kloning

White Spot Syndrome Virus (WSSV) dapat menyebabkan kematian kumulatif hingga 100% pada budidaya udang dalam waktu 10 hari. Protein VP28 merupakan salah satu protein amplop WSSV yang terlibat dalam penempelan dan penetrasi virus ke dalam sel udang. Vaksinasi menggunakan bakteri yang mengekspresikan protein VP28 WSSV diharapkan dapat mengatasi infeksi WSSV pada udang windu (Penaeus monodon). Tujuan penelitian ini adalah mengklon kerangka baca terbuka gen pengkode VP28 dalam vektor ekspresi pMSP3535VA pada Escherichia coli DH5a. pMSP3535VA adalah vektor shuttle yang dapat melakukan replikasi di E. coli dan bakteri gram positif. Klon E. coli yang mengandung pMSP3535VA dengan kerangka baca terbuka gen pengkode VP28 sebagai DNA sisipan telah berhasil diperoleh. Plasmid rekombinan dikarakterisasi dengan analisis migrasi, pemotongan dengan enzim restriksi BamHI dan EcoRI, analisis dengan Polymerase Chain Reaction (PCR), dan penentuan urutan nukleotida DNA sisipan. Karakterisasi plasmid rekombinan mengunakan analisis migrasi menunjukkan pMSP3535VA telah disisipi DNA sisipan, pemotongan plasmid rekombinan menunjukkan plasmid mengandung DNA sisipan berukuran 619 pasangan basa (pb), analisis PCR menunjukkan satu pita berukuran 643 pb, dan urutan nukleotida DNA sisipan memiliki tigkat kesamaan 100% dengan urutan nukleotida kerangka baca gen pengkode VP28.

Deskripsi Alternatif :

The cumulative mortality of White Spot Syndrome Virus (WSSV) infection in shrimp aquaculture may reach 100% within 10 days. VP28 is one of WSSV envelope proteins which is involved in attachment and penetration of virus into shrimp cells. The use of bacteria which expressed VP28 protein as a vaccine is expected to solve WSSV infection in black tiger shrimp (Penaeus monodon). The goal of this experiment is to clone open reading frame (ORF) of VP28 gene into an expression vector pMSP3535VA in Escherichia coli DH5a. pMSP3535VA is a shuttle vector which is able to propagate in E. coli and gram positive bacteria. E. coli clone that contains pMSP3535VA with VP28 ORF inserted was obtained. The recombinant plasmid was characterized using migration analysis, cleavage by restriction enzyme BamHI and EcoRI, Polymerase Chain Reaction (PCR) analysis, and nucleotide sequencing of the inserted DNA. The result of the characterization using migration analysis showed that pMSP3535VA had been ligated with insert DNA, cleavage recombinant plasmid showed that size of insert DNA was 615 base pairs (bps), PCR analysis showed one band with 643 bps in size, and nucleotides of the inserted DNA had 100% homology with VP28 ORF.

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