Cloning And Expression Analysis Of Terminal Complement Components Of Cyprinus Carpio Haematopteru

Membrane attack complex (MAC)can run through cell membrane and leading to lysis of targetcell.MAC is composed by terminal complement components(TCC),such as C6,C7,C8α,C8β and C9.This essay cloned and analyzed the TCC gene: C8α,C8β and C9of common carp (Cyprinus carpiohaematopterus Temminck et Schlegel) and analyzed the expression of C8α,C8β and C9gene in differentstissues and early development stage by RT-PCR. The results are as follows:According to the homolog sequence of vertebrates' TCC family,Four pairs of oligonucleotide primerssynthesised by primer primer5.0software were used to amplification intermediate fragment of geneC8α,C8β and C9by RT-PCR. The full-length cDNA sepuence obtained by RT-PCR and RACE of C8αhad2066nt, encoding595amino acids. And the sepuence similarities with Zerbafish(Danio rerio),trout(Oncorhynchus mykiss)and sweetfish (Plecoglossus altivelis)were72%,53%and51%respectively. Thefull-length cDNA sepuence of C8β had1994nt,encoding573amino acids.And the similarities of sepuencewith Zerbafish(Danio rerio),trou(tOncorhynchus mykiss), Fugu(Takifugu rubripes) and Japanese flounder(Paralichthys olivaceus) were75%,61%,58%56%. The full-length cDNA sepuence of C9had2028,encoding675amino acids.And the similarities of sepuence with grass carp(Ctenopharyngodon idella),Zerbafish(Danio rerio),trou(tOncorhynchus mykiss)and Japanese flounder (Paralichthys olivaceus)77%,75%,49%and49%. There were28conserved Cys residues which were highly conserved in vertebratesTCC family includes:C6,C7, C8α,C8β and C9,they all had the TSP,LDLRA,MACPF and the EGF domain.The C8α,C8β and C9gene expression analysis in differents tissues and early development stage byRT-PCR revealed that C8α,C8β and C9were expression in all detected tissues,includinggills,muscle,spleen,kidney,head kidney,heart,brain,intestine and blood.It indicate C8α,C8β and C9geneConstitutive expression.In early development stage. C8α and C8β gene had a similar expression. Duringblastula stage and gastrula stage, the expression level of C8α and C8β gene were low, while increased inthe stage of tail-bud and coordinated during in blastula stage,there was on C9gene expression in blastulastage, gastrula stage and stage of tail-bud.C9gene was detected in the stage of heart beating and the embryo pro-hatching. the expression level decreased in the embryo just hatching, but increased in2dayhatching. C9gene expression was not detected after2day hatching.