PENGKLONAN GEN PENYANDI VIRAL PROTEIN 15 (VP-15) WSSV DAN APLIKASINYA SEBAGAI VAKSIN REKOMBINAN PADA UDANG WINDU

Andi Parenrengi, Sri Redjeki Hesti Mulyaningrum, Andi Tenriulo, Agus Nawang

Abstract


Infeksi white spot syndrome virus (WSSV) dapat menyebabkan kematian massal pada budidaya udang windu Penaeus monodon di Indonesia. Infeksi yang terjadi secara sistematis tersebut disebabkan oleh peran gen nucleocapsid viral protein (VP-15). Upaya pengembangan gen VP-15 WSSV untuk menginduksi respons imun dan menetralisasi terhadap infeksi WSSV pada udang windu perlu dilakukan. Penelitian ini bertujuan untuk mengisolasi dan merekombinasikan gen penyandi VP-15 WSSV sebagai vaksin dsRNA, serta menganalisis aplikasinya pada udang windu. Gen VP-15 diisolasi dari udang windu yang terinfeksi WSSV, dikloning ke dalam suatu vektor dan ditransformasikan ke sel kompeten (bakteri Escheria coli DH5a). Plasmid diisolasi untuk mengonfirmasi insert region gen VP-15 melalui sekuensing nukleotida. Pembuatan vaksin rekombinan dilakukan secara in-vitro menggunakan kit MEGAscript RNAi dan diaplikasikan ke udang windu melalui metode injeksi dengan dosis tunggal 0,2 µg dan kontrol (tanpa injeksi vaksin). Hewan uji yang digunakan berukuran panjang 14,75±3,17 g dan bobot 11,64±0,76 cm; serta dipelihara pada wadah bak fiber volume 250 L dengan kepadatan 10 ekor/bak. Hasil penelitian menunjukkan bahwa gen penyandi VP-15 telah diisolasi dari udang windu dan vaksin rekombinan telah dihasilkan secara in-vitro. Analisis sekuens nukleotida memperlihatkan bahwa sisipan gen DNA VP-15 sebesar 253 bp dan menunjukkan kemiripan yang tinggi (99%) pada GenBank. Penggunaan vaksin rekombinan dsRNA dengan dosis 0,2 µg memperlihatkan sintasan udang windu yang dapat mencapai 40,0% dibandingkan dengan kontrol hanya 3,3% (peningkatan 36,7%). Gambaran histopatologi pada jaringan hepatopankreas udang windu pada perlakuan kontrol menunjukkan adanya kerusakan inti sel, akibat infeksi WSSV. Gene VP-15 berpotensi sebagai bahan vaksin rekombinan dsRNA dalam mencegah infeksi WSSV.

Infection of white spot syndrome virus (WSSV) causes bulk mortalities of tiger shrimp Penaeus monodon cultured in Indonesia. The nucleocapsid viral protein-15 (VP-15) is strongly suspected to be responsible for the systemic infection of WSSV. The development of VP-15 WSSV gene for inducing the immune response to and neutralize WSSV infection of tiger shrimp is vitally needed. The aim of this study was to isolate and clone the gene encoding VP-15 WSSV as dsRNA vaccine and assess the vaccine application to tiger shrimp. VP-15 gene was isolated from the genomic DNA of infected tiger shrimps, cloned into the vector, and transformed into competent cells (Escheria coli DH5a). The plasmid was isolated to confirm the insert region gene of VP-15 by the nucleotide sequence. Production of dsRNA vaccine was performed by in-vitro using MEGAscript RNAi kit and applied to tiger shrimp through muscular injection at a single dosage of 0.2 µg and without dsRNA as a control treatment. The average size of tiger shrimps used was 14.75±3.17 g in weight and 11.64±0.76 cm in length and stocked in 250 L fiber tank at 10 ind./tank. The results of the study showed the VP-15 gene was successfully isolated from the tiger shrimps and the recombinant vaccine was produced by in-vitro. The analysis of nucleotide sequence showed that the inserted DNA was 253 bp and showed a high similarity (99%) with VP-15 gene deposited in the GenBank. The application of dsRNA vaccine showed that the dosage of 0.2 ¼g resulted in the survival rate of 40.0% compared with without dsRNA (control) of 3.3% (36.7% increment). Hepatopancreas histology indicated obvious damages to cell nucleus in the un-vaccinated tiger shrimp caused by the virus infection. We suggest that the VP-15 gene is a very promising dsRNA recombinant vaccine against WSSV infection.




Keywords


kloning; gen VP-15 WSSV; vaksin rekombinan; dsRNA; udang windu; cloning; VP-15 WSSV; recombinant vaccine; dsRNA; tiger shrimp

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DOI: http://dx.doi.org/10.15578/jra.13.1.2018.57-65


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