Molecular Cloning, Sequence Analysis And Expression Of Yu Complement Component C9Gene

Complement system, which is able to destroy pathogens either by causing their lysis directlyor by recruiting phagocytic cells indirectly, is one of the most powerful effector systems involvedin the body’s defenses and inflammation. C9is a component of the membrane attack complex(MAC), participates in the final stage of the complement cascade which lyses foreign organisms bydisrupting the integrity of their cell membranes. However, the expression of ayu C9(aC9)following bacterial infection has not been well described. In the present study, a2125nt length ayuC9cDNA sequence encoding592amino acids was determined. A signal peptide is deposited inthe N-terminal22residues. Domain search revealed that aC9contains four domain types of TSP1(thrombospondin type-1), LDLa (low-density lipoprotein receptor class A), EGF-like (epidermalgrowth factor like) and MACPF (MAC/perforin). The deduced amino acid sequence of aC9showed the highest56.8%identity to rainbow trout, and40.9%to53.8%identity to other teleosts.Phylogenetic tree analysis showed that the C9of fish and mammals formed two species-specificclusters. RT-PCR analysis demonstrated that the mRNA of aC9gene was expressed in allexamined organs of healthy fish except kidney, brain and heart, with the highest level inhepatopancreas. The quantitative RT-PCR analysis showed that aC9transcripts were significantlyup-regulated in hepatopancreas at4h post Listonella anguillarum infection reaching2.65foldscompared with the control group. The relative expression of aC9gene reached the peak at16hpost injection, and up-regulated to31.97folds of control level. After that, the aC9transcriptsreduced but still significantly higher than control group (19.79folds). With the up-regulation ofaC9transcripts in hepatopancreas, an obvious up-regulated tendency of C9as time post injection inserum was observed, and peaked at36h post infection (5.8folds). The western blotting analysis ofthe serum aC9digested by PNGase F revealed that the mature aC9in serum is a type ofglycoprotein. Our results suggested that aC9may play an important role in fish immune responseof anti-bacteria.