KLONING GEN PUTATIVE CLEAVAGE PROTEIN 1 (PCP-1) PADA UDANG VANAME (Litopenaeus vannamei) YANG TERSERANG INFECTIOUS MYONECROSIS VIRUS

Hessy Novita, Agus Sunarto, Septyan Andriyanto

Abstract


Penanggulangan penyakit ikan dapat dilakukan dengan cara meningkatkan kekebalan tubuh ikan melalui program vaksinasi. Namun vaksinasi tidak tepat untuk udang, karena udang tidak mempunyai immunological memory seperti ikan. Oleh karena itu, perlindungan udang terhadap serangan penyakit viral dengan menggunakan RNA interference (RNAi). Teknologi RNAi digunakan untuk menghalangi (interfere) proses replikasi infectious myonecrosis virus (IMNV) pada udang vaname dengan cara menon-aktifkan gen putative cleavage protein 1 (PCP-1), yang berfungsi dalam pembentukan capsid dan proses transkripsi RNA IMNV. Penelitian ini bertujuan untuk melakukan kloning gen putative cleavage protein 1 dalam rangka perakitan teknologi RNAi untuk pengendalian penyakit IMNV pada udang vaname. Tahapan penelitian meliputi koleksi sampel, isolasi RNA, sintesis cDNA, amplifikasi PCR, purifikasi DNA, transformasi, isolasi plasmid, serta sekuensing dan analisis data. Hasil isolasi plasmid cDNA PCP-1 memperlihatkan semua koloni bakteri terseleksi ternyata membawa plasmid hasil insersi DNA gen PCP–1, hasil sekuen dengan nilai homologinya mencapai 100% dan 99% yang dibandingkan dengan sekuen di Genebank. Hasil penelitian menunjukkan bahwa kloning gen putative cleavage protein 1 (PCP-1) dari udang vaname yang terserang Infectious Myonecrosis Virus berhasil dikloning yang nantinya digunakan untuk perakitan RNAi.

The prevention of fish diseases can be done by increasing of the fish immune through vaccination programs. However, the vaccination can not be done for the shrimp,due to the absence of  immunological memory. Therefore, the protection of shrimp against viral diseases was done by using of RNA interference (RNAi). RNAi technology is used to interfere infectious myonecrosis virus (IMNV) replication process on white shrimp by disabling of putative cleavage protein 1 (PCP-1)gene, which functions in capsid formation and RNA transcription process. The study was conducted to perform putative cleavage protein I gene cloning in the framework of RNAi technologies for IMNV disease control in white shrimp. This study consisted of sample collection, RNA isolation, cDNA synthesis, PCR amplification, DNA purification, transformation, plasmid isolation, sequencing and data analysis.The cDNA PCP-1 plasmid isolation showeds that all selected bacterial colonies appeared lead insertion plasmid DNA PCP-1 gene with100% and 99% sequence homology compared to sequence in Genebank. The results  exhibited that the putative cleavage protein 1 (PCP-1) gene cloning from infectedwhite shrimp of IMNV was completed successfully and used for the framework of RNAi.


Keywords


Litopenaeus vannamei; IMNV; gen PCP-1; kloning; Litopenaeus vannamei; IMNV; PCP-1 gene; cloning; Litopenaeus vannamei; IMNV; PCP-1 gene; cloning

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DOI: http://dx.doi.org/10.15578/ma.11.1.2016.27-33


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