Pengembangan Vaksin DNA Penyandi Glikoprotein Virus KHV (Koi Herpesvirus) Menggunakan Isolat Lokal

Abstract

Sekuen gen glikoprotein yang berukuran 1,8 kbp dan berasal dari ORF 25 virus KHV berpotensi untuk dijadikan gen target dalam konstruksi vaksin DNA. Berdasarkan hasil analisis sekuens seperti diungkapkan di atas, fragmen DNA tersebut merupakan gen GP25. Setelah GP25 diligasi ke vektor pGEMT Easy (pT, berukuran 3 kbp), maka ukuran plasmid menjadi sekitar 5 kbp. Digesti plasmid pTGP25 menggunakan enzim Sal I menghasilkan fragmen GP25 yang berukuran sekitar 1,8 kbp. Berdasarkan hasil sekuensing dan analisis kemiripan (similarity) dengan menggunakan software BLAST diketahui bahwa GP25 dari virus KHV asal Indonesia memiliki kemiripan yang tinggi (99%) dengan GP25 KHV asal Jepang, Amerika Serikat dan Israel. Selanjutnya, pAct-D6 yang berukuran sekitar 8,5 kbp dipotong dengan enzim Sal I untuk membuang gen D6 (berukuran sekitar 1,6 kbp). Setelah pAct diligasi dengan gen GP25 dihasilkan plasmid pAct-GP25 dengan ukuran sekitar 8,8 kbp. Karena situs restriksi yang digunakan hanya satu jenis, maka kemungkinan arah ligasi bisa 2 macam. Untuk menentukan plasmid dengan arah ligasi yang diinginkan, maka dilakukan analisis PCR. Produk PCR untuk plasmid dengan arah ligasi yang benar memiliki ukuran sekitar 2,1 kbp (nomor 6, 17 dan 20). Konstruksi pActGP25 ini merupakan plasmid DNA sirkuler yang digunakan sebagai vaksin, yang disingkat dengan nama GP25. Untuk menguji aktivitas vaksin DNA GP25 yang telah dikonstruksi maka dilakukan uji ekspresi gen β-aktin yang digunakan sebagai promoter vaksin. Uji ekspresi gen dilakukan terhadap konstruksi pActGFP (representasi promoter β-aktin) dan pActGP25. Uji aktivitas promoter β-aktin dilakukan dengan menginjeksikan secara intramuscular pActGFP ke juvenil ikan mas ukuran 10-15 g/ekor. Konsentrasi DNA plasmid yang diinjeksikan adalah 12,5 μg/100μL fosfat buffer salin (PBS). Total RNA diekstraksi dari otot yang diinjeksi, insang, limpa dan ginjal ikan mas yang diinjeksi dengan pAct-GFP pada saat 24 jam pasca injeksi (hari pertama) dan 1 minggu pasca injeksi. Sementara itu untuk pAct-GP25, total RNA diekstraksi dari jaringan/organ yang sama dengan pada pAct-GFP, tetapi dilakukan pada 24 jam, 2 minggu dan hari 4 minggu setelah injeksi. Ekstraksi RNA dilakukan menggunakan Isogen (Nippon Gen, Japan). Sintesis cDNA dilakukan dengan menggunakan kit Ready-To-Go You-Prime First-Strand Beads (Amersham Pharmacia Biotech, USA).

The aim of this research was to develop DNA vaccine of Koi Herpesvirus in carp. Glycoprotein gene originated from ORF 25 KHV virus that is 1.8 kbp in size, is a potential gene in DNA vaccine construction. The sequencing and similarity analysis of the gene using BLAST software showed that this gene is a GP25 gene and was found to be very similar (99%) to GP25 of KHV virus from the USA and Israel. When this GP25 gene was ligated with pGEMT Easy (pT, 3 kbp in size) vector, plasmid size was increasing up to 5 kb. Following this, a digestion of pT-GP25 plasmid using Sal I enzyme was applied resulted in a GP25 fragment (1.8 kbp). Subsequently, pAct-D6 with a size of 8.5 kbp was also cut using the same enzyme to eleminate a D6 gene with a size of 1.6 kbp. GP25 gene was then ligated with pAct resulted a pAct-GP25 plasmid with a size of 8.8 kbp. As there was only one restriction site applied, ligation can be occurred in two different directions. Thus, in order to determine the plasmid with the desired direction PCR analysis was carried out, and plasmid with the right direction was the one that sized approximately 2.1 kbp (no. 6, 17 and 20). High level of GFP gene expression was detected in all tissues analyzed, i.e. kidney, gil, spleen, and muscle 24 hours after injection which indicated that β-actin promoter of Japanese medaka could be active in common carp. Although not as high as on 24 hours, expression was still detected until day 7 and disappeared after day 28 post injection indicating that pAct-GFP can be remained and β-actin promoter of Japanese medaka can be active in muscle, gills and kidney at least a week after injection. The result of RT-PCR analysis on pAct-GP25 injected common carp showed that GP25 gene expression can be detected 14 days after injection in all tissues observed. The expression level differences between GFP and GP25 genes possibly related to the primer sensitivity on annealing process to cDNA template and the size of PCR targeted DNA fragment, as smaller PCR targeted product will be easier to be amplified than a bigger one. The length of PCR targeted DNA with GP25 primer was 1.8 kbp whereas GFP gene was 0.6 kbp.The challenge test on common carp results showed that all unvaccinated and negative control (not infected with KHV) was alive until the end of the experiment. Whereas, the survival of fish vaccinated with a dose of 12.5 was ranged from 60.9 and 96.7%. In relation to the activity of β-actin promoter that was used in this DNA vaccine, gene expression was occured 24 hours after vaccination and remained until 2 weeks. The control fish that was not injected by GP25 showed no GP25 expression either in the observed tissues or organs and resulted in a lower survival, i.e. 23.3%. The challenge test results has indicated that the fish vaccinated with GP25 at doses of 7.5 and 12.5 μg/100μL were able to produce KHV glycoprotein in their body which would be recognized as an antigen that further would activate the fish immune response. In the challenge test, the active immune response was then represented in a higher survival. This result also showed that GP25 is an immunogenic and its expression could enhance common carp immunity on KHV infection.