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Cloning, Sequencing Analysis and Eukaryotic Expression of Full Length Growth Hormone cDNA of Carassius Auratus Gibelio Var

Author: PeiShuLi
Tutor: LiuZhongHu
School: Henan Agricultural University
Course: Molecular Biology
Keywords: Carassius auratus gibelio var growth hormone(GH) gene cloning Sequence analysis recombinant plasmid eukaryotic expression
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 106
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Abstract


China is a big country in aquaculture. Freshwater fishery has an important role in meat food. Carassius auratus gibelio var, natural triploid gynogenesis carp, is a local fish in Henan province, which grows fast, displays delicious taste and other performance. To promote breeding and product, support sustainable development of the fish, and provide safe product for food market, it is nessary that develop new fish growth promoters to increase growth and ensure food safety. On the other hand, growth hormone(GH)denatures and disappears quickly and could not be accumulated in the fish body. It is suggested that application of recombinant growth hormone from native animal could be the best selection for meeting the demand.Fish GH is peptide hormone secreted by the pituitary, associated with fish growth, development, metabolism, the regulation of appetite and osmotic pressure, and it is widely used in study involved in fish Endocrinology, feeding and food conversion efficiency, growth, and so on. However, it is difficult to get a lot of natural growth hormone, which limited its functions and applied research.GH gene cDNA of Carassius auratus gibelio va was cloned and sequenced in this study, the protein sequence of the GH was derived and the homology of evolution was compared and analysed with other fish. In the future we can research the growth Pattern from the molecular level .In the eukaryotic expression study, the primers were designed according to the fish GH full-length sequence obtained from the previous resulte , specific primer was designed at both ends of the open reading frame, use RT-PCR to identify the size of the 633bp. The analysis result of complete sequence shows it is correct.The vector has been constructed and transinfected into E. coli DH5a, using PCR to initial screening of recombinant ,using EcoRI, SnaBI double digested further identified, Recombinant will be named PPIC9K-GH it will provide recombinant for the next step study after sequencing.Recombinant will be electric shocks transformed into Pichia GS115. Use the MD and G418 screening the positive bacteria has high resistance. Yeast genomes PCR to identify carriers have been integrated into the reorganization of yeast. And the positive yeast integrated was sequenced .The positive strains will be inoculated and be methanol-induced expression, the expressed protein was showen obviously at the 22 kDa band in SDS-Page gel electrophoresis, this result revealed that recombinant GH in Pichia GS115 was expressed and secreted. To screening positive high expression strains, will provide a solid foundation for the further study.

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