Research On Characterization And Preliminary Hemolysis Function Of Complement Component 3 In Sinonovacula Constricta

Razor clam(S.constricta)is an important economic shellfish in China.Mollusk only can resist antigen with innate immune system.The complement system,as one of the innate immune system,plays a crucial role in process of resistance antigen.To date,the study of complement C3 in mollusk focused on sequence and structure analysis.In this paper,three C3 genes of razor clam(ScC3,ScC3-2,ScC3-3)had been obtained and analysed on gene structure,mRNA expression,and hemolysis function.1.Three partial sequences of ScC3 genes were obtained from transcriptome of razor clam.PCR was used to verify the partial sequences,RACE-PCR was used to obtain the full lenth cDNA sequences.The length of three ScC3 genes is 6950 bp,6294bp,and 5965 bp,encoding these as amino acids 1752,1741,and 1701,respectively.Amino acids sequences of three C3 genes were different,which showed 67% identical between ScC3 and ScC3-2,35% identical between ScC3 and ScC3-3,38% identical between ScC3-2 and ScC3-3.However,the secondary structure of three C3 proteins was quite similary with 3D structure.The structure of three ScC3 genes contained eight conserved domains that belong to C3,but in ScC3-3 didn’t found Thioester and α-γ junction structure.The phylogenetic tree indicated that all of three ScC3 are classical complement C3,among them,ScC3 is more similary with ScC3-2.2.The three ScC3 mRNA expression pattern were detected in healthy tissues and development stages of razor clam with qRT-PCR.The results showed that ScC3、ScC3-2 and ScC3-3 mRNA were widely expressed,and had significant difference during every developmental stages.After razor calm challenged with Vibrio Parahaemolyticus,the results for ScC3,up-regulated in hemolymph at 8h and up-regulated in liver at 72 h,for ScC3-2,up-regulated in hemolymph at 8h,and for ScC3-3,up-regulated in hemolymph at 24 h,up-regulated in liver at 72 h.In addition,the results of razor calm challenged with Micrococcus lysodeikticus showed that all of the ScC3,ScC3-2 and ScC3-3 are down-regulated in hemolymph at 24 h and up-regulated in liver at 4h.Those results indicated that all of ScC3,ScC3-2 and ScC3-3 could participate in process of immune response as a valid immune gene,but performed different function for three ScC3 genes.3.The several physical factors were used to explore the optimum reaction conditions of hemolysis in razor clam.The results showed that hemolysis depend on Ca2+,Mg2+ and alkalescence.Hemolysis could be effected by temperature,time,SCP concentration(optimum condition: 4:6)amd incubation pattern(optimum condition: low volatility for incubation).It indicated that the condition of SCP hemolysis is different to hemolysis condition of mammalian.It suggested that mollusk might exist special immune mechanism.Moreover,some biochemical factors were used to study the activity of hemolysis,showed that hemolysis could be activated by LPS,zymosan,FLA and PGN,also could inhibited by PMSF and methylamine.4.The specific antibody(ant-ScC3,ant-ScC3-2 and ant-ScC3-3)were selected to perform inhibition activity of three razor clam C3 proteins in hemolysis.Then,the antibody was verified specificity by immunoprecipitation test.The results showed that ScC3 and ScC3-2 could perform hemolysis.Western Blot and Laser confocal imaging were used to locate three ScC3 proteins in process of hemolysis,turned out that the three ScC3 located on surface of cells and bacteria.It indicated that the ScC3 and ScC3-2 are quite similar in their function,but ScC3-3 is different.Thus,we could concluded that ScC3 is a heat-sensitive protein and have serine protease activity protein,work on surface of cells in hemolysis process.5.The downstream genes in complement system could not be searched from transcriptome data.The method of homologous recombination was used to design primers for structuring recombinant plasmid(pEGFP-N1)of three ScC3.Then in RAW264.7 cells,the recombinant plasmid was overexpression tested.It showed that three ScC3 could effectively active mouse complement system in vitro.It indicated that the complement system of razor clam is typical cascade reaction,which need to be actived by activator.