Characterization and expression of recombinant mouse garoupa (Cromileptes altivelis) growth hormone and insulin-like growth factor genes

• Main Author: Ag. Muhammad Sagaf Abu Bakar
• Format: Thesis
• Language: English; English
• Published: 2007
• Subjects: QL614-639.8 Fishes
• Online Access: https://eprints.ums.edu.my/id/eprint/6799/1/24%20PAGES.pdf; https://eprints.ums.edu.my/id/eprint/6799/2/FULLTEXT.pdf; https://eprints.ums.edu.my/id/eprint/6799/

The growth hormone (GH) is an important polypeptide produced in the anterior region of the pituitaty gland which is required for normal growth and development of vertebrates. The insulin-like growth factor 1 (IGF-I), on the other hand is regulated by GH at the transcriptional level in the liver. The mouse garoupa is an important cultured marine finfish . One of the major challenges in rearing this fish is its slow growth rate compared to other commercial grouper species. A pre-pro GH and a partiallGF-1 complementary DNA (cDNA) was isolated using a modified rapid amplification of cDNA ends (RA CE) method, which is a homology-base RACE-PCR (HRACE-PCR). The GH cDNA consisted of a 920 bp fragment having a 615 bp open reading frame (ORF) encoding a peptide of 204 amino acids. The partial IGF-1 cDNA consisted of a 496 bp encoding 150 amino acids containing the B, C, A, D and a partial E domain. For expression and production of recombinant mouse garoupa GH (rmGH), a cDNA segment encoding the mature peptide of the preGH was inserted into an Escherichia coli expression vector, pTrcHis (Invitrogen) which is controlled by the trc promoter. Induction under 1.0 mM isopropyl l3-thiogalactosidase (lPTG) at 37°C resulted in the overexpression of a 23 kDa protein corresponding to the predicted size based on amino acid sequence. An in-gel Western blot detection for histidine tagged protein furthermore showed that the 23 kDa band contains a 6X hisitdine tag. The mGH was expressed as inclusion bodies and was further isolated, purified and refolded. To asses this problem, an economical, scalable and rapid process has been developed based on detergent dialysis. The use of the p TrcG His construct resulted in the production of an estimated 19-21 % of the E. coli cellular protein as mGH. A preliminary bioassay to study conducted on the effects of oral administration of mGH to tilapia fingerlings administrated twice a week at concentrations of 1 ng/g, 10 ng/g and 100 ng/g body weight resulted in fishes administered with the highest dosage gaining a 50% (P<0.05) increase in weight and 10% (P<0.05) in length when compared to the control over a period of six weeks. These results indicated that the mGH was biologically active and refolded.