IV. HASIL DAN PEMBAHASAN
4.8 Ekspresi dan Analisis Protein rGH
Berdasarkan hasil analisis SDS-PAGE (Gambar 17), terdapat pita yang muncul pada posisi 25 kDa yang diduga merupakan protein rGH ikan mas. Berdasarkan hasil tersebut, bisa disimpulkan bahwa protein rGH ikan mas telah berhasil diproduksi. Dari 200 ml media 2xYT dapat dihasilkan 0,93 g pellet bakteri yang mengandung rGH. Dari pellet bakteri yang diproduksi, diperkirakan ada sebanyak 10,51% protein rGH dari total protein yang dihasilkan (diprediksi dengan menggunakan program Totallab TL 120). Cheng (1995) menyatakan bahwa rGH yang diproduksi oleh E. coli sekitar 8-10% dari total protein yang dihasilkan.
23 Gambar 17. Hasil SDS-PAGE protein rGH; M= marker, 1= protein dari bakteri E. coli BL21 (DE3) yang membawa pCold I tanpa insersi, 2= protein dari bakteri E. coli BL21 (DE3) yang membawa plasmid C-mCcGH; tanda panah menunjukkan protein rGH ikan mas; angka di sebelah kiri menunjukkan ukuran marker
Berdasarkan Gambar 17, dapat diketahui bahwa bakteri E. coli BL21 (DE3) yang membawa plasmid C-mCcGH menghasilkan rGH yang berukuran sekitar 25 kDa (ditunjukkan dengan tanda panah). Berbeda dengan protein yang dihasilkan dari bakteri E. coli BL21 (DE3) yang membawa pCold I tanpa insersi, tampak tidak ada protein pada ukuran 25 kDa. Tetapi, protein lain yang diproduksi relatif sama. Berat molekul protein yang dihasilkan lebih besar dari rGH yang diduga berdasarkan asumsi yaitu 21 kDa karena adanya penambahan dari His Tag yang terdapat di dalam vektor pCold I (Chan et al. 2003; Roberts et al. 2004). Penebalan pita protein pada posisi selain 25 kDa yang tidak terdapat pada protein dari bakteri E. coli BL21 (DE3) yang membawa pCold I tanpa insersi diduga merupakan struktur lain GH.
4.9 Uji Bioaktivitas rGH Ikan Mas
Bioaktivitas rGH ikan mas yang diproduksi dapat diketahui dengan membandingkan pertumbuhan mutlak ikan mas yang disuntikkan rGH ikan mas dan ikan mas yang disuntikkan dengan pCold I tanpa insersi. Dari Gambar 18 dapat terlihat bahwa ikan mas yang disuntikkan dengan rGH ikan mas memiliki
M 1 2 58 - 30 - 25 - 17 - kDa ◄ rGH
24 pertumbuhan yang lebih besar bila dibandingkan dengan ikan mas yang disuntikkan dengan pCold I tanpa insersi. Hal ini menandakan bahwa rGH ikan mas yang diproduksi aktif dan dapat memacu pertumbuhan ikan.
Gambar 18. Bobot ikan mas yang disuntikkan dengan pCold I tanpa insersi (pCold I) dan rGH ikan mas (mCcGH) yang dipelihara selama 8
minggu. Dosis penyuntikkan 1 μg GH/10 μl PBS/g bobot
tubuh/minggu selama 4 minggu
Pada akhir penelitian, penyuntikan rGH ikan mas meningkatkan pertumbuhan sebesar 106,56% bila dibandingkan dengan ikan mas yang disuntikkan dengan pCold I tanpa insersi. Hasil ini lebih tinggi dibandingkan dengan yang dilaporkan pada ikan sebelah, 24% (Jeh et al. 1998), juvenil ikan gilthead seabream, 29-33% (Ben-Atia et al. 1999), dan ikan mas koki, 43% (Promdonkoy et al. 2004), tetapi lebih rendah dibandingkan dengan yang dilaporkan oleh Mahmoud et al. (1998) pada ikan mas yaitu 120% dan Acosta et al. (2007) pada ikan nila yaitu 171%. Perbedaan peningkatan pertumbuhan tersebut diduga terkait dengan jenis dan ukuran ikan uji, sumber GH yang digunakan, dan metode pemberian GH.
Pertumbuhan mutlak ikan yang disuntikkan dengan rGH sebesar 12,98±0,56 g. Lebih tinggi bila dibandingkan dengan ikan yang disuntikkan dengan pCold I
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 1 2 3 4 5 6 7 8 Bobo t Ikan (g) Minggu ke- pCold I mCcGH
25 tanpa insersi yaitu 6,28±4,69 g (Gambar 19). Walaupun uji statistik tidak menunjukkan perbedaan yang nyata antar perlakuan (P>0,05) (Lampiran 6), tetapi berdasarkan trend pertumbuhan yang didapatkan, terdapat indikasi bahwa rGH yang dihasilkan aktif secara biologi dan dapat digunakan untuk memacu pertumbuhan organisme akuakultur sehingga dapat mempercepat masa pemeliharaan untuk mendapatkan ukuran ikan yang diinginkan. Kedepannya, untuk kemudahan dalam mengaplikasikannya, pemberian rGH dapat dilakukan melalui perendaman larva atau juvenil ikan dalam larutan rGH (Acosta et al.
2007) maupun mencampurkannya dengan pakan yang akan diberikan (Ben-Atia et al. 1999; Promdonkoy et al. 2004).
Gambar 19. Pertumbuhan mutlak ikan mas yang disuntikkan dengan protein dari pCold I tanpa insersi (pCold I) dan rGH ikan mas (mCcGH)
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 Pert umbu ha n mut lak (g) Perlakuan pCold I mCcGH
V. KESIMPULAN DAN SARAN
5.1 Kesimpulan
Protein rGH ikan mas dapat diproduksi dengan menggunakan vektor pCold I di dalam bakteri E. coli dan terindikasi memiliki bioaktivitas dalam memacu pertumbuhan ikan mas.
5.2 Saran
Protein rGH ikan mas yang dihasilkan dapat digunakan oleh petani ikan untuk memacu pertumbuhan ikan lambat. Lebih jauh, perlu pengembangan dalam metode, dosis, dan waktu pemberian yang tepat agar lebih efisien dalam aplikasinya.
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33 Lampiran 1. Sekuens cDNA GH ikan mas berdasarkan Genbank M27000
LOCUS CYIGH 1164 bp mRNA linear VRT 08-AUG-1994 DEFINITION Cyprinus carpio growth hormone (GH) mRNA, complete
cds. ACCESSION M27000
VERSION M27000.1 GI:529027 KEYWORDS growth hormone.
SOURCE Cyprinus carpio (common carp)
ORGANISM Cyprinus carpio
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Actinopterygii; Neopterygii; Teleostei; Ostariophysi; Cypriniformes; Cyprinidae; Cyprinus. REFERENCE 1 (bases 1 to 1164)
AUTHORS Koren,Y., Sarid,S., Ber,R. and Daniel,V.
TITLE Carp growth hormone: molecular cloning and sequencing of cDNA
JOURNAL Gene 77 (2), 309-315 (1989)
PUBMED 2753359
COMMENT On Aug 9, 1994 this sequence version replaced gi:341578.
Original source text: Cyprinus carpio pituitary gland cDNA to mRNA. FEATURES Location/Qualifiers source 1..1164 /organism="Cyprinus carpio" /mol_type="mRNA" /db_xref="taxon:7962" /tissue_type="pituitary gland" gene 1..1164 /gene="GH" CDS 37..669 /gene="GH" /codon_start=1 /product="growth hormone" /protein_id="AAA49208.1" /db_xref="GI:529028" /translation="MARVLVLLSVVLVSLLVNQGRASDNQRLFN NAVIRVQHLHQLAAKMINDFEDSLLPEERRQLSKIFPLSFCNSD YIEAPAGKDETQKSSMLKLLRISFHLIESWEFPSQSLSGTVSNS LTVGNPNQLTEKLADLKMGISVLIQACLDGQPNMDDNDSLPLPF EDFYLTMGENNLRESFRLLACFKKDMHKVETYLRVANCRRSLDS NCTL" sig_peptide 37..102 /gene="GH" mat_peptide 103..666 /gene="GH" /product="growth hormone" polyA_signal 1142..1147 /gene="GH" polyA_site 1164 /gene="GH" ORIGIN 1 aactaagcctgcaagagtttgtctaccctgagcgaaatggctagagtattagtgctattg 61 tcggtggtgctggttagtttgttggtaaaccaggggagagcatcagacaaccagcggctc 121 ttcaataatgcagtcattcgtgtacaacacctgcaccagctggctgcaaaaatgattaac 181 gactttgaggacagcctgttgcctgaggaacgcagacagctgagtaaaatcttccctctg
34 241 tctttctgcaattctgactacattgaggcgcctgctggaaaagatgaaacacagaagagc 301 tctatgctgaagcttcttcgcatctcttttcacctcattgagtcctgggagttcccaagc 361 cagtccctgagcggaaccgtctcaaacagcctgaccgtagggaaccccaaccagctcact 421 gagaagctggccgacttgaaaatgggcatcagtgtgctcatccaggcatgtctcgatggt 481 caaccaaacatggatgataacgactccttgccgctgccttttgaggacttctacttgacc 541 atgggggagaacaacctcagagagagctttcgtctgctggcttgcttcaagaaggacatg 601 cacaaagtcgagacctacttgagggttgcaaattgcaggagatccctggattccaactgc 661 accctgtagatggcaccggtgtattgttagtcaatgcctgtaacacatttgtgctttgct 721 gcaaatataagaccagtttacagtctggtcttatatgtgcaggaaatgtcaaccagcatg 781 cctaggtctgtgttttcttttttccctcccatatttaaacattacctattgtatttattc 841 ttctcatttgggagtgtctcataaatttaaaaccgttcctttaaaacgtaagggatggat 901 ctggaacatttcacagtggtgtctaagcaatttatggcaatattttaaaatgtggccaaa 961 ttgaccttagtcaaagtgctgacaatatgttaaaaaaagggctaaagatcagtgttacgt 1021 ggaaattgtaatttaaatcggatgtgttcactcttcggtgtatgcatgttaacatttgtc 1081 tcatatattatgctcttattattaactcatcgtatcctcttcaagcgctgtgtctttctc 1141 tattaaagttttaaattgcatcca
35 Lampiran 2. Prediksi sinyal peptida CDS GH ikan mas
>Sequence
SignalP-NN result:
# data
>Sequence length = 70
# Measure Position Value Cutoff signal peptide? max. C 23 0.964 0.32 YES
max. Y 23 0.889 0.33 YES max. S 3 0.981 0.87 YES mean S 1-22 0.899 0.48 YES D 1-22 0.894 0.43 YES
# Most likely cleavage site between pos. 22 and 23: GRA-SD
SignalP-HMM result:
36 # data
>Sequence
Prediction: Signal peptide
Signal peptide probability: 1.000 Signal anchor probability: 0.000
37 Lampiran 3. Hasil sekuensing CcGH-1
39 Lampiran 4. Hasil sekuensing CcGH-48
41 Lampiran 5. Hasil pensejajaran CcGH-1 dan CcGH-48 dengan GH mature
Mature Mas.txt 0 - - - 0 ccGH 1-pCold.gnu 1 - - -TG G GTATA ACAC C CATGA ATCACA A AGTGCATCATCATCATCATCATATCGA AG GTA 57 ccGH 48-pCold.gnu 1 A A AG G G GTATA-CAC C-ATGA ATCACA A AGTGCATCATCATCATCATCATATCGA AG GTA 58 Mature Mas.txt 1 - - - -TCAGACA AC CAGCG GCTCT TCA ATA ATGCAGT 32 ccGH 1-pCold.gnu 58 G GCATATG GAGCTCG GTAC C CTCGAG GCTCAGACA AC CAGCG GCTCT TCA ATA ATGCAGT 117 ccGH 48-pCold.gnu 59 G GCATATG G- - - -CTCAGACA AC CAGCG GCTCT TCA ATA ATGCAGT 100 Mature Mas.txt 33 CAT TCGTGTACA ACAC CTGCAC CAGCTG GCTGCA A A A ATGAT TA ACGACT T TGAG GACAG 92 ccGH 1-pCold.gnu 118 CAT TCGTGTACA ACAC CTGCAC CAGCTG GCTGCA A A A ATGAT TA ACGACT T TGAG GACAG 177 ccGH 48-pCold.gnu 101 CAT TCGTGTACA ACAC CTGCAC CAGCTG GCTGCA A A A ATGAT TA ACGACT T TGAG GACAG 160 Mature Mas.txt 93 C CTGT TGC CTGAG GA ACGCAGACAGCTGAGTA A A ATCT TC C CTCTGTCT T TCTGCA AT TC 152 ccGH 1-pCold.gnu 178 C CTGT TGC CTGAG GA ACGCAGACAGCTGAGTA A A ATCT TC C CTCTGTCT T TCTGCA AT TC 237 ccGH 48-pCold.gnu 161 C CTGT TGC CTGAG GA ACGCAGACAGCTGAGTA A A ATCT TC C CTCTGTCT T TCTGCA AT TC 220 Mature Mas.txt 153 TGACTACAT TGAG GCGC CTGCTG GA A A AGATGA A ACACAGA AGAGCTCTATGCTGA AGCT 212 ccGH 1-pCold.gnu 238 TGACTACAT TGAG GCGC CTGCTG GA A A AGATGA A ACACAGA AGAGCTCTATGCTGA AGCT 297 ccGH 48-pCold.gnu 221 TGACTACAT TGAG GCGC CTGCTG GA A A AGATGA A ACACAGA AGAGCTCTATGCTGA AGCT 280 Mature Mas.txt 213 TCT TCGCATCTCT T T TCAC CTCAT TGAGTC CTG G GAGT TC C CA AGC CAGTC C CTGAGCG G 272 ccGH 1-pCold.gnu 298 TCT TCGCATCTCT T T TCAC CTCAT TGAGTC CTG G GAGT TC C CA AGC CAGTC C CTGAGCG G 357 ccGH 48-pCold.gnu 281 TCT TCGCATCTCT T T TCAC CTCAT TGAGTC CTG G GAGT TC C CA AGC CAGTC C CTGAGCG G 340 Mature Mas.txt 273 A AC CGTCTCA A ACAGC CTGAC CGTAG G GA AC C C CA AC CAGCTCACTGAGA AGCTG GC CGA 332 ccGH 1-pCold.gnu 358 A AC CGTCTCA A ACAGC CTGAC CGTAG G GA AC C C CA AC CAGCTCACTGAGA AGCTG GC CGA 417 ccGH 48-pCold.gnu 341 A AC CGTCTCA A ACAGC CTGAC CGTAG G GA AC C C CA AC CAGCTCACTGAGA AGCTG GC CGA 400 Mature Mas.txt 333 CT TGA A A ATG G GCATCAGTGTGCTCATC CAG GCATGTCTCGATG GTCA AC CA A ACATG GA 392 ccGH 1-pCold.gnu 418 CT TGA A A ATG G GCATCAGTGTGCTCATC CAG GCATGTCTCGATG GTCA AC CA A ACATG GA 477 ccGH 48-pCold.gnu 401 CT TGA A A ATG G GCATCAGTGTGCTCATC CAG GCATGTCTCGATG GTCA AC CA A ACATG GA 460 Mature Mas.txt 393 TGATA ACGACTC CT TGC CGCTGC CT T T TGAG GACT TCTACT TGAC CATG G G G GAGA ACA A 452 ccGH 1-pCold.gnu 478 TGATA ATGACTC CT TGC CGCTGC CT T T TGAG GACT TCTACT TGAC CATG G G G GAGA ACA A 537 ccGH 48-pCold.gnu 461 TGATA ATGACTC CT TGC CGCTGC CT T T TGAG GACT TCTACT TGAC CATG G G G GAGA ACA A 520 Mature Mas.txt 453 C CTCAGAGAGAGCT T TCGTCTGCTG GCT TGCT TCA AGA AG GACATGCACA A AGTCGAGAC 512 ccGH 1-pCold.gnu 538 C CTCAGAGAGAGCT T TCGTCTGCTG GCT TGCT TCA AGA AG GACATGCACA A AGTCGAGAC 597 ccGH 48-pCold.gnu 521 C CTCAGAGAGAGCT T TCGTCTGCTG GCT TGCT TCA AGA AG GACATGCACA A AGTCGAGAC 580 Mature Mas.txt 513 CTACT TGAG G GT TGCA A AT TGCAG GAGATC C CTG GAT TC CA ACTGCAC C CTG- - - 564 ccGH 1-pCold.gnu 598 CTACT TGAG G GT TGCA A AT TGCAG GAGATC C CTG GAT TC CA ACTGCAC C CTGTAG GTCA A 657 ccGH 48-pCold.gnu 581 CTACT TGAG G GT TGCA A AT TGCAG GAGATC C CTG GAT TC CA ACTGCAC C CTGTAG GTCA A 640 Mature Mas.txt 564 - - - 564 ccGH 1-pCold.gnu 658 TCACTAGTGA AT TCGCG GC CGC CTGCAG GTCGAC CTGCAGTCTAGATAG GTA ATCTCTGC 717 ccGH 48-pCold.gnu 641 TCACTAGTGA AT TCGCG GC CGC CTGCAG GTCGAC CTGCAGTCTAGATAG GTA ATCTCTGC 700 Mature Mas.txt 564 - - - 564 ccGH 1-pCold.gnu 718 T TA A A AGCACAGA ATCTA AGATC C CTGC CAT T TG GCG G G GAT T T T T T T T TAT T TGT T T T T 777 ccGH 48-pCold.gnu 701 T TA A A AGCACAGA ATCTA AGATC C CTGC CAT T TG GCG G G GAT T T T T T T- -AT T TGT T T T- 757 Mature Mas.txt 564 - - - 564 ccGH 1-pCold.gnu 778 CAG GA A ATA A A ATA ATCGATCGCGTA A A- - - 805 ccGH 48-pCold.gnu 758 CAG GA A ATA A ATA ATCGATCGCGTA ATA A A ATCTAT TAT TAT T T T T TGTGA AGA AT 813
42 Lampiran 6. Hasil uji statistik pertumbuhan mutlak (G)
t-Test: Paired Two Sample for Means
pCold I mCcGH
Mean 6.282222 12.97677 Variance 22.02066 0.3192
Observations 2 2
Pearson Correlation -1 Hypothesized Mean Difference 0
df 1 t Stat -1.80073 P(T<=t) one-tail 0.161359 t Critical one-tail 6.313752 P(T<=t) two-tail 0.322719 t Critical two-tail 12.7062