Molecular Cloning,Recombinant Expression Analysis Of C-type Lectin From Scapharca Broughtonii

The ark shell,Scapharca broughtonii,belonging to Mollusca,Bivalve,Pterimorphia,Arcoida,Arcidae,is an important marine economic bivalve.It has become one of the most popular farming mollusks in China due to its high economic value.The resources of S.broughtonii decreases deeply due to overfishing,pollution and the occurring of disease because of unhealthy aquaculture.The main consentration of the study in the ark shell is on basic biology and aquaculture technique.The research of s.broughtonii is short of molecular immunity in recent years.The research of molecular mechanism related to immunity plays an impormant part in improving and enriching the chief of immunology data in the art shell.On the base of 454 transcriptome sequencing,we cloned four c-type lectin named Sb-Lec1?Sb-Lec2?Sb-Lec3 and Sb-Lec4 by expressed sequence tag(EST)and rapid amplification of cDNA ends(RACE)techniques.The study analysed the tissue distribution of the four genes and the expression mechanism after Vibrio anguillarum expresure.In the meanwhile,the Sb-Lec2 protein was expressed successfully and detected the bacteria coagulation activity in escherichia coli and vibrio anguillarum,staphylococcus aureus.This research includes the following contents:1.The full-length cDNA of C-type lectin(Sb-Lec1)was cloned using RACE(rapid amplification of cDNA ends)method from Scapharca broughtonii.Sequence analysis showed that the full-length of the gene is 700 bp.The open reading frame(ORF)is 504 bp and encodes a polypeptide of 167 amino acids,including a signal peptide of 23 amino acids,one CRD motif of 129 amino acids and six cysteines involved in the formation of disulfide bond.The predicted protein molecular weight is 19.11 kDa,with a theory isoelectric point of 4.74.The results of multiple sequences alignment and phylogeny analysis show that the identity of Sb-Lec1 shared with other species such as four conserved Cys.All the sequences were clustered into two main branches after phylogenetic analysis in which all C-type lectin of molluscs clustered together,while the C-type lectin of vertebrate formed the another separate cluster.Quantitative Real-time PCR(qRT-PCR)was used to assess the mRNA distribution in all tested tissues,including hemocytes,foot,adductor muscle,mantle,gill,and hepatopancreas.The highest expression level was observed in hepatopancreas.This study also analyzed the mRNA expression changes of Sb-Lec1 post Vibrio anguillarum exposure.Results showed that the mRNA expression profile of Sb-Lec1 were significant up-regulated compared with the control group in each detected tissue(P < 0.05).2.The full-length cDNA of Sb-Lec2 is 756 bp.The ORF is 489 bp and encodes a polypeptide of 162 amino acids,including one CRD motif of 135 amino acids and six cysteines.The predicted protein molecular weight is 19.11 kDa,with a theory isoelectric point of 4.74,which belongs to secreted protein.All the sequences were clustered into two main branches after phylogenetic analysis in which all C-type lectin of molluscs clustered together,while the C-type lectin of fish and mammals formed the another separate cluster,which indicated that the deduced polypeptide of Sb-Lec2 had the characteristics of the C-type lectin family.Quantitative Real-time PCR(qRT-PCR)was used to assess the mRNA distribution in all tested tissues.The highest expression level was observed in hepatopancreas.The results of the mRNA expression changes of Sb-Lec2 post Vibrio anguillarum showed that the mRNA expression profile of Sb-Lec2 were significant up-regulated compared with the control group in each detected tissue.The recombinant protein of Sb-Lec2 expressed in the form of inclusion body after prokaryotic gene recombinant protein expression.After purification,dialysis renaturation,the protein showed antimicrobial activity in Vibrio anguillarum,Escherichia coli,Staphylococcus aureus with no Ca2+ dependence and no mannose binding activity.3.The full-length cDNA of Sb-Lec3 and Sb-Lec4 is 559 bp,603 bp.The ORF of Sb-Lec3 and Sb-Lec4 is 492 bp and and 360 bp,which encodes a polypeptide of 163 amino acids and 119 bp.The predicted protein molecular weight of Sb-Lec3 is 18.96 kDa,with a theory isoelectric point of 5.01,while the predicted protein molecular weight of Sb-Lec4 is 12.37 kDa,with a theory isoelectric point of 5.80.The two genes has signal peptide which indicates they are secreted protein.These genes have similarity with other speciese,for example,conservative cysteine.All the sequences were clustered into two main branches after phylogenetic analysis in which all C-type lectin of molluscs clustered together,while the C-type lectin of other species formed the another separate cluster.The results of qRT-PCR indicated that the expression of the genes was distributed in all tested tissues.The highest expression level was observed in hepatopancreas.This study also analyzed the mRNA expression changes of Sb-Lec3 post Vibrio anguillarum exposure.Results showed that in every organization,the phenomenon of the expression quantity rise before reducing.