Production and bioactivity test of recombinant protein common carp growth hormone

Abstract

<p>This study aimed to produce recombinant growth hormone <em>(r</em>GH) protein of common carp (<em>Cyprinus carpio</em>) and evaluate its bioactivity. DNA fragment encoding mature GH protein of common carp (<em>mCc</em>GH) was amplified by polymerase chain reaction (PCR) method and PCR products were then ligated into pCold I to generate pCold I-<em>mCc</em>GH protein expression vector. <em>Escherichia coli </em>BL21 (DE3) harboring pCold I-<em>mCc</em>GH was cultured in the 2xYT medium at 15 °C for 24 hours and protein production was induced by isopropyl-beta-thio galactopyranoside (IPTG). The inclusion bodies containing rGH protein from <em>E. coli </em>transformants were isolated by sonication method and analyzed by SDS-PAGE. The result showed that rGH with molecular weight of about 25 kDa was obtained. Common carp juveniles with average body weight of 5.2±0.4 g were intramuscularly injected once a week for 4 weeks with rGH protein solution from 1 μg bacterial cells per gram fish body weight. The result showed that juveniles fish injected with rGH grew 106.56% higher than control. This result indicated that rGH produced in <em>E. coli </em>BL21 possessed biological activity and it may be useful to improve growth of aquaculture species.</p> <p>Key words: growth hormone, recombinant protein, common carp</p> <p> </p> <p>ABSTRAK</p> <p>Penelitian ini bertujuan menghasilkan protein rekombinan hormon pertumbuhan (<em>growth hormone</em>, GH) dari ikan mas (<em>Cyprinus carpio</em>) dan menguji bioaktivitasnya. Fragmen DNA penyandi protein matang (<em>mature</em>) GH ikan mas (<em>mCc</em>GH) diamplifikasi dengan menggunakan metode PCR dan hasilnya kemudian diligasi ke dalam pCold-I untuk menghasilkan konstruksi vektor ekspresi pCold-I-<em>mCc</em>GH. Plasmid pCold-I-<em>mCc</em>GH ditransformasi ke bakteri <em>Escherichia coli</em> BL21 (DE3), dikultur dalam media 2xYT cair pada suhu 15°C selama 24 jam dan produksi protein diinduksi dengan menggunakan isopropyl-beta-thio galactopyranoside (IPTG). Badan inklusi yang mengandung protein rekombinan GH (rGH) dari bakteri <em>E. coli</em> transforman diisolasi menggunakan metode sonikasi dan dianalisis dengan menggunakan SDS-PAGE. Hasil penelitian menunjukkan bahwa rGH dengan bobot molekul sekitar 25 kDa berhasil diproduksi. Benih ikan mas dengan bobot rata-rata 5,15±0,4 g diinjeksi secara intramuskular satu kali per minggu selama 4 minggu dengan larutan rGH hasil ekstraksi dari 1 µg pelet bakteri/g bobot ikan. Benih yang disuntik dengan rGH tumbuh sekitar 100% lebih cepat bila dibandingkan dengan kontrol yang tidak diinjeksi rGH. Hasil ini mengindikasikan bahwa rGH yang diproduksi dalam bakteri <em>E. coli</em> memiliki bioaktivitas dan dapat bermanfaat untuk memacu pertumbuhan spesies ikan-ikan budidaya.</p> <p>Kata kunci: hormon pertumbuhan, protein rekombinan, ikan mas</p>