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Cloning and Expression of Growth Hormone Gene in Cobia (Rachycentron Canadus)

Author: HaoYu
Tutor: LiuChuWu
School: Guangdong Ocean University
Course: Marine biology
Keywords: Rachycentron canadus Growth hormone Gene cloning Escherichia coli Expression
CLC: Q786
Type: Master's thesis
Year: 2011
Downloads: 13
Quote: 0
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Abstract


Growth hormone (GH) is a member of GH/PRL/SL family that regulates normal growth and reproduction in fish. DNA recombination technique offered the feasibility to produce GH gene recombinant individual fish and realized growth-promoting by adding exogenous growth hormone in fish. In this study, the pituitary growth hormone (GH) gene of cobia was cloned, characterized and expressed. The tissue distributions are investigated using Real-time PCR method.We designed a pair of degenerate primers according to several kinds of GH genes from different Perciformes fishes. Using this pair of primer, we amplified cDNA fragments of GH gene from cobia pituitary cDNA. Subsequently, we designed one pair of primers for amplification of 5’ and 3’sequences of GH gene by homology cloning. Then, we obtained the 5’ and 3’ UTR of cobia GH genes and the full length cDNA of GH. The full-length of GH DNA contains 1790bp nucleotides with a 77bp 5’ untranslated region (UTR), 170bp of 3’ UTR and an open reading frame (ORF) of 1533bp, containing five exons and four introns, encoding a protein of 204 amino acids comprising a 22 amino acids signal peptide and a 182 amino acids mature protein with a theoretical molecular weight(MW) of 23.17kDa and an estimated isoelectric point(IP) of 6.43.The result of homology analysis showed that GH gene in cobia has the lower identity to GH in mammals and nonmammalian tetraposds, which was 40%. But the identity of GH in cobia and in Perciformes fishes was higher than 80%.GH is an important component of the somatotropic axis (GH–IGF-1), which controls growth and development in fish,Expression analysis of GH by revealed that cobia GH was mainly expressed in pituitary, and weakly expressed in ovary, liver, Stomach,brain, spleen, kidney, heart, muscle and gill.The cDNA encoding sequence of GH was sequenenced and subcloned to expression vector pET-28a after digesting with NdeⅠand XhoⅠ(named as pET-CoGH), and expressed in E.Coli BL21(DE3). SDS-PAGE analysis showed that the molecular weight of expressed fusion protein is 23kDa. The western-blot analysis showed that the His antibody could specifically combiant with recombinant protein pET-CoGH. In this study, we optimized the expression conditions of GH gene from the host bacteria, induction temperature, induction time, concentration of IPTG, Experiments showed that GH gene was not expressed in the host bacteria BL21, but expressed in BL21(DE3). The recombinant protein largely expressed at 37℃, with 1mmol / L IPTG when induced 4 h, the recombinant protein was partly soluble when induced at 25℃.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering) > Gene expression
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