Klonasi dan analisis sekuen gen penyandi protein Capsid Iridovirus isolat Indonesia

HANDAYANI, Christina Retna, Dr. Rarastoeti Pratiwi, M.Sc

2008 | Tesis | S2 Bioteknologi

Iridovirus diketahui sebagai agen penyebab penyakit sistemik serius pada ikan air tawar dan ikan laut. Di Indonesia, kematian lebih dari 80% dilaporkan terjadi pada kerapu lumpur (Epinephelus coioides) yang dipelihara pada jaring tancap di Sumatera Utara. Gen penyandi protein capsid iridovirus ini diduga mempunyai potensi untuk pengembangan vaksin rekombinan dan metode deteksi. Tujuan dari penelitian ini adalah mendapatkan klon pembawa gen penyandi protein capsid iridovirus yang dilakukan dengan pendekatan klonasi langsung produk PCR dan melakukan analisis sekuen gen tersebut. Gen penyandi protein capsid iridovirus isolat Jepara dan Bali berhasil diklonasi pada pBSKS dan disekuensing. Sekuen komplit dari open reading frame (ORF) gen penyandi protein capsid iridovirus isolat Jepara berukuran 1362 bp yang menyandi 453 asam amino. Kedua isolat iridovirus yang dihasilkan dari penelitian ini termasuk dalam genus Megalocyctivirus dan mempunyai similaritas 99,8% pada level nukleotida serta 99,4% pada level asam amino. Isolat Jepara dan Bali mempunyai similaritas 99,6-99,9% terhadap DGIV, ISKNV, MCIV, dan ALIV. Berdasarkan hasil prediksi epitop sel T dan sel B, protein capsid iridovirus diduga bersifat imunogenik, sehingga plasmid rekombinan yang membawa gen penyandi protein capsid iridovirus isolat Jepara dan Bali dapat digunakan untuk pengembangan vaksin rekombinan dan metode deteksi.

Iridoviruses are recognized as causative agents of serious systemic diseases both freshwater and marine fishes. The virus has been reported infect estuary grouper (Epinephelus coioides) reared in net-cage in North Sumatera, with the mortality of more than 80%. Gene encode major capsid protein of this virus was predicted has potency for development of recombinant vaccine and detection method. The purposes of this research are to clone gene encode major capsid protein of iridovirus with direct PCR product cloning approach and to analyse the sequences. Gene encoded major capsid protein from Jepara and Bali isolates were successfully cloned into pBSKS and sequenced. The full length of Open reading frame (ORF) of gene encode iridovirus major capsid protein from Jepara isolate sized 1362 bp which encode 453 amino acids. Both iridovirus isolates are classified into genus Megalocyctivirus and their similarities are 99.8% in nucleotide level and 99.4% in amino acid level. Jepara and Bali isolates have similarity 99.6-99.9% against DGIV, ISKNV, MCIV, and ALIV. According to T and B cell epitope prediction results, major capsid protein of iridovirus is predicted to be immunogenic, so that recombinant plasmid encoded iridovirus major capsid protein of Jepara and Bali isolates can be used for the development of recombinant vaccine and detection method.

Kata Kunci : Infeksi Iridovirus,Pengendalian,Klonasi,Protein Capsid, cloning, major capsid protein, iridovirus, Epinephelus coioides