The Study On Cloning And Prokaryoti Expression Of Pseudosciaena Crocea Insulin-like Growth Factor-â… 

Insulin-like growth factors(IGFs), are single-chain polypeptide hormones,including IGF-Ⅰand IGF-Ⅱ, whose structure and function are similar to insulin,IGFs have fifty percent homology with proinsulin. IGFs play a important role instimulating growth and differentiation, IGFs also have important physiologicalfunction in osmoregulation, reproduction, life cycle, sclerotin, Immune response andmetabolism. It is difficult to collect IGFs of biological activity, because most of IGFsare bound to IGF-binding proteins and its content is low.As the research of fish IGFs system, it is very important to get IGFs of biologicalactivity. This research studied the prokaryotic expression of IGFs in the E.coliexpression systems at the basis of cloning Pseudosciaena crocea IGF-Ⅰ.The insulin-like growth factor (IGF-Ⅰ) gene of Pseudosciaena crocea was clonedfrom liver by RT-PCR and RACE technique. All the length of the nucleotide sequenceis2027bp, including5’UTR (57bp),3’UTR (1409bp) and ORF (561bp). Nucleotidesequence analysis showed that ORF encoded186amino acids that is composed ofsignal peptide, mature peptide (B, C, A, D) and E domain. Signal peptide and Edomain are cut off when the peptide is processed. The molecular weight of maturepeptide is7500Da and its pI is7.76. There are three disulfide bonds formed by sixcysteines in B and A domains. The disulfide bonds comparing the amino acidsequence of IGF-Ⅰof P. crocea with those of other species, the sequence conservationof A and B domains is high, while low in C and D domains. E domains in IGF-Ⅰof P.crocea is belonged to IGF-ⅠEa-4subtype. Analysis of nucleotide sequence showedthat IGF-ⅠcDNA of P. crocea is similar to other fishes which shared sequencehomology of99.47%with Sciaenops ocellatus,97.68%with Epinephelus coioides,97.50%with Sparus aurata and95.90%with Paralichthys olivaceus.Prokaryotic expression systems of the pET-28a/IGF-Ⅰwere constructed andused to express IGF-Ⅰ. The results of SDS-PAGE showed that the IGF-Ⅰhad amolecular weight of about24kDa.