Gene Clonging And Eucaryotic Expression Of Turbot

Turbot, Scophthalmus maximus, is an important species in aquaculture and it contributes enormously to the economic development of coastal provinces in China. However, with the farming environment worsening, it had caused catastrophic losses to aquaculture. The intensive efforts had been accelerated for development of better health management strategies and characterization of original immune efforts because of the outbreak of diseases. Although it is still not very clear on the pathogen infection of the turbot, the immune mechanism plays a key role in controlling outbreak of disease in turbot. Thus, understanding the immune mechanism of turbot could contribute to develop strategies for management of the disease and provide guidance about the disease control for other marine fishes.Antimicrobial peptides (AMPs) are regarded as important components of the host innate immune system and play crucial roles in host defense against microbial invasion. Based on the expression and recombination of antimicrobial peptides genes, we can find an attractive way of fishery breeding and disease control of marine fishes. And insulin-like growth factor-I (IGF-I) is a kind of active peptide that can promote many types of cells to grow. It plays an important regulatory role in the proliferation, differentiation, cell apoptosis and tumor genesis. Moreover, recombinant expression of antimicrobial peptides in yeast provides us an opportunity to explore the recombinant strains as additive feedstuff of fishery. The study mainly focuses on following research contents:1. Molecular cloning and expression analysis of turbot IGF-I gene; Quantitative real time PCR (QRT-PCR) demonstrated that IGF-I transcripts were highly abundant in liver, abundant in eye, skin, brain, gill, gonad, spleen; less abundant in muscle, heart, intestine and head kidney. The level of IGF-I mRNA in embryos gradually increases during embryogenesis after fertilization. The high temperature stimulation research indicated that IGF-Ⅰhas the inhibitory action in liver. And Expression analysis of IGF-Ⅰin starvation turbot liver decreased significantly on 16th day.2. That five secretion expression vectors of yeast were constructed with gene recombination, and five intracellular expression vectors also constructed. With western blot, it was indicated that four recombinant yeasts, pPICZαA-HIP, pPICZαA-HGP, pPICZαA-HP and pPICZαA-IP were made. The recombinant protein was detected with western blot, and it was shown that the expression of protein of pPICZαA-HIP and pPICZαA-HGP was increasing with time by methyl alcohol induced. It was shown that there was some antibacterial activity with pPICZαA-HIP, pPICZαA-HGP and pPICZαA-HP. And the activity of pPICZαA-HP was the most. And GFP was found with pPICZαA-HGP in the yeast and around.3. That five fluorescence protein fusion expression vectors of cell were constructed, eGFP-HN, eGFP-IN, eGFP-HIN, eGFP-GN and eGFP-MN. With GFP detected, it suggested that there were 3 recombinant cell lines, eGFP-HIN, eGFP-GN and eGFP-MN, made with gene recombination. And it was shown different GFP distribution in them.In summary, the IGF-Ⅰgene was cloned, and its expression was detected with QRT-PCR. It was confirmed that several recombinant yeasts were made and the recombinant yeast expression system of turbot was established. And 3 recombinant cell lines were also made. It has laid the foundation for later study on function and action of gene, gene interation, new feed additive development and so on.