Klonasi dan analissi sekuen gen penyandi protein membran virus herpes koi

DARIUS, Dr. Ir. Ngadiman, M.Si

2008 | Tesis | S2 Bioteknologi

Virus herpes koi (koi herpesvirus/KHV) merupakan virus yang menyerang ikan mas (Cyprinus carpio) dan ikan koi (Cyprinus carpio koi), sangat menular dan menyebabkan kematian secara masal. Saat ini belum ada vaksin yang dapat mencegah serangan KHV. Penelitian ini bertujuan untuk memperoleh klon yang membawa gen penyandi protein membran (PM) KHV isolat Indonesia, yang merupakan langkah awal dalam tahapan untuk mendapatkan protein sebagai kandidat vaksin. KHV isolat indonesia diisolasi dari insang ikan mas yang didapatkan dari danau Toba, Sumatera Utara. Gen penyandi PM KHV diamplifikasi menggunakan PCR (polymerase chain reaction) dengan primer spesifik yang dirancang berdasarkan data GenBank. Hasil amplifikasi diligasikan dengan vaktor pBluescript II KS +/- dan ditransformasikan ke dalam Escherichia coli DH5(alfa) . Plasmid rekombinan yang diisolasi dari transforman, dikonfirmasi menggunakan pemotongan dengan enzim restriksi, kemudian dilakukan pengurutan (sequensing). Urutan nukleotida gen PM KHV dianalisis dengan menggunakan program Genetyx, Cromas Pro, BLAST, ClustalW2, IEDB, IMF bioinformatics, dan BepiPred. Hasil penelitian menunjukkan bahwa gen PM KHV isolat Indonesia telah diklon kedalam E.Coli DH5(alfa) dan berukuran 837 bp. Hasil analisis Multiple sequence alignment menunjukkan bahwa homologi gen PM KHV isolat Indonesia dengan gen-gen PM KHV isolat Israel (AB178324) dan isolat Jepang (AB178537) yang ada pada basis data GenBank adalah 99%. Analisis kandungan GC dan titik isoelektrik berturut-turut adalah 68,74% dan 6,33. Analisis terhadap asam amino mengungkapkan bahwa PM KHV isolat Indonesia mempunyai 17 epitop sel T pola IAd; 4 epitop sel T pola IEd; dan 8 epitop sel T pola Rothbard/Taylor. Sementara analisis epitop sel B menunjukkan bahwa PM KHV ini mempunyai 7 epitop sel B. Berdasarkan hasil tersebut PM KHV isolat Indonesia berpotensi sebagai imunogen.

Koi herpesvirus (KHV) is a viral disease attacking common carp (Cyprinus carpio) and koi (Cyprinus carpio koi), very contagious and causing mass mortality. In this time there is no vaccine able to prevent KHV’s attack. This research was aimed to clone membrane protein (MP) gene of Indonesia’s KHV isolate. It is the early step to produce protein as a vaccine candidate. Indonesian KHV was isolated from carper gills got from lake of Toba, North Sumatra. Gene of PM KHV was amplified using PCR ( polymerase chain reaction ) with specific primers which were designed according to Genbank data. The product of amplification was ligated to pBluescriptII KS +/- vector and transformed into Escherichia coli DH5(alfa) .The presence of insert in recombinant plasmid which was isolated from transforman was confirmed by digesting with restriction enzyme, then the recombinant plasmid was sequenced. Sequence of MP KHV gene subsequently analysed by programs of Genetyx, Cromas Pros, BLAST, Clustalw2, IEDB, IMF Bioinformatics, and BepiPred. The results showed that gene of MP KHV was successfully cloned into E.coli DH5(alfa). The length of MP KHV encoding gene is 837 bp. Analysis of Multiple sequence alignment indicated that MP KHV gene homology of Indonesian isolate with Israel’s isolate (AB178324) and Japan’s isolate (AB178537) are 99%. The results of GC content and isoelectrict point analysis of MP KHV Indonesian isolate are 68,74% and 6,33 respectively. Analysis of deduced Amino Acid sequence revealed that MP KHV Indonesian isolate have 17 T-cell epitopes of IAd pattern; 4 T-cell epitopes of IEd pattern; and 8 T-cell epitopes of Rothbard / taylor pattern. Whereas analysis of B-cell epitope indicate that MP KHV have 7 cell epitopes B. Based on these results, it can be theoretically concluded that MP KHV of Indonesian isolate has some putative immunogens.

Kata Kunci : Klonasi,Protein membran (PM),Sekuen,Epitop sel T dan B, KHV, membrane protein (MP) , PCR, sequence, T and B cell epitopes