Preliminary Functional Analysis Of Collectin And Transferrin In Grass Carp(Ctenopharyngodon Idella)

In this study, we had preliminary screened liver and kidney grass carp (Ctenopharyngodon idella) cDNA library which had been constructed. In the library the full-length cDNA sequences of grass carp collectin (GQ141696) gene had been cloned by sequencing and characteristics identifying. The full length sequence of grass carp collectin was 1128 bp, contained a 5'untranslated region of 229 bp and a 3' untranslated region of 104 bp. The open reading frame of grass carp collectin was 795 bp which could code a 264 amino acids polypeptide, including a terminal codon. Phylogenetic analyses showed that grass carp collectin shared the highest homology with that of zebrafish (Danio rerio). To understand the function of grass carp collectin, we expressed and purified the recombinant protein (PCRD) that comprised carbohydrate recognition domain (CRD). Agglutination of Aeromonas hydrophila and Staphylococcus aureus etc. and sugars inhibition experiments showed that:galactose, glucose, mannose and maltose could inhibit the agglutination of Aeromonas hydrophila. Maltose could lower the agglutination of Staphylococcus aureus, whereas peptidoglycan and glucose inhibited it well. In addition, the activity of grass carp collectin could not dependent on Ca2+Tf(transferrin, Tf) was aβ-globin of non-heme combination with iron, which is connected by a short hinge region between the N-terminal and C-terminal homology domain. Grass carp transferrin has four iron ion binding sites in each domain, which combine with four Fe3+ respectively. Transfetrin has diverse functions that it not only participates in the transportation and metabolism of organism iron ions, but also plays key effect in cellular respiration,cellular growth and proliferation,antimicrobial bactericidal bioactivity and related to innate immunity. In order to understand the molecular mechanism of binding of fish transferrin and iron ion, and the function of N-terminal and C-terminal homology domain. In this study, cDNA sequences were cloned containing these two domains respectively according to the sequence characteristics of the full length of grass carp transferrin gene (EU790874). we expressed and purified the recombinant protein (PTf1, PTf2). Then we detected the capacity of binding with iron ion for recombinant proteins. Waveband scan drawing of sample showed that recombinant protein can combine with iron ion,causing the changes of the absorption peak. Binding experiments in vitro showed that the capacity of iron binding of two domains were different, the capacity of iron combination of PTf2 was stronger than PTf1.