Cloning And Expression Analysis Of Complement C9 And Perforin Genes In Grass Carp Ctenopharyngoden Idellus

C9 is the ninth member of complement components,creating the membrane attack complex(MAC) in lytic pathway.Perforin is a soluble,pore forming cytolytic glycoprotein synthesized in CTL and NK cells and sequestered into secretory cytotoxic granules.Both molecules have been shown to undergo polymerization to form circular lesions,and they share some structural and functional similarities.In the present study,the complement C9(gcC9) and perforin(gcPFP) genes were cloned in grass carp Ctenopharyngodon idella.GcC9 gene with a full cDNA length of 2123 bp has been cloned using RACE-PCR,which contains an open reading frame(ORF) of 1950 bp coding for 650 aa.Domain search revealed that gcC9 contains a LDL receptor domain,an EGF precursor domain,a MACPF domain and two TSP domain.GcC9 gene consists of 11 exons with 10 introns,spacing over 7003 bp of genomic sequence.Analysis of gcC9 promoter region-of 822 bp revealed the presence of a TATA box and some putative transcription factors such as C/EBP,HSF,NF-AT,CHOP-C,HNF-3B,GATA-2, IK-2,EVI-1,AP-1,CP2 and OCT-1 binding site.By RT-PCR and quantitative real-time PCR analysis,the transcription of gcC9 was detected in eggs,and the peak value occurred at 48 h post fertilization(hpf).Using pQE-30 expression vector, recombinant gcC9 protein was obtained and used to immunize rabbits for generating polyclonal antibody.The distribution of gcC9 transcripts and protein in different organs of healthy grass carp were examined by RT-PCR and Western blotting analysis,respectively. The gcC9 had a constitutive expression in all examined organs,and had a significantly high expression in liver,about 72 kDa was detected in liver,head kidney, renal kidney,spleen,brain,heart,gill,blood,muscle,thymus,skin and intestine.In response to F.columnare injection,the gcC9 mRNA increase was observed in liver at day 1 and in spleen at day 7 post treatment;and in response to Poly I:C treatment,the gcC9 transcription increase was observed in spleen at day 1,in liver.and head kidney at day 3 post treatment.The gcPFP-1 and gcPFP-2 were cloned by using degeneracy primers,the gcPFP-1 with the full cDNA length of 2514 bp which contains an ORF of 1764 bp coding for 588 aa,and the gcPFP-2 full cDNA length is 2254 bp,including a 1737 bp ORF which encodes 579 aa.The amino acid sequence of gcPFP-1 is 58%identical to that of gcPFP-2,and the two gcPFPs all contain MACPF and C2 domain which are found in perforin.The gcPFP-1 and gcPFP-2 all consist of 4 exons with 3 introns, spacing over 4090 bp and 3059bp of genomic sequence,respectively.The distribution of gcPFP-1 and gcPFP-2 transcripts had a similar pattern in all detected organs,the transcription of gcPFPs were detected at 6 hpf during development,with the peak value detected at 48 hpf,followed by decline.The polyclonal antibodies to gcPFP-1 and gcPFP-2 show immunological cross-reactivity.GcPFP-1 and gcPFP-2 are all about 68 kDa,and had a constitutive expression in all examined organs.In response to F.columnare injection,gcPFP-1 and gcPFP-2 mRNA increase was observed in spleen and head kidney at day 7 post treatment;in response to PolyI:C injection,gcPFP-1 and gcPFP-2 transcription increase was observed in spleen at day 1,in intestine and head kidney at day 3,post treatment,gcPFP-1 transcription increase but gcPFP-2 decline was observed in liver at day 3 post two treatment.The gcC9 and gcPFP are structurally related;gcPFP has approximately 20% amino acid identity with gcC9,and they all have the MACPF and EGFP domains. PFP and C9 share two domains that fulfill roles essential to their overall functions, namely those providing for lipid insertion and polymerization.The present study suggested that fish possess multiple isoforms of PFP,and gene duplication events may have given rise to diverse gene isotypes in the PFP family.This suggestion was supported by the genomic information of zebrafish,pufferfish and stickleback.