Cloning And Function Analysis Of Caspase Gene In Sea Cumber, Apostichopus Japonicus

Apoptosis,It is an active cell suicide process through growth, differentiation,developmental and pathological changes.caspase(Cysteine aspartic acid specific protease)，itplays an important role in cell apoptosis、differentiation、proliferation、necrosis andinflammation process.The full-length cDNA sequence of Ajcasp1were firstly cloned byRACE technique from coelomocyte of sea cucumber Apostichopus japonicus according tothe partial sequence previously obtained, named as Ajcasp1(GeneBank No.: KC972624).Bioinformatic analysis:The full-length cDNA of Ajcasp1was2100bp,has a5’UTR of181bpand3’UTR of782bp with an open reading frame(ORF) of1137bp encoding334aminoacids.Sequence analysis showed that AjCASP1has caspase family’s classical domaincontains N-terminal prodomains(CARD caspase-recruitment domain),P20large subunit,P10small subunit, the coserved “QACXG” motif and a histidine active site with some activesites,chemical binding sites,proteolytic cleavage sites and polypeptide binding sites.Domain analysis found that the N-terminal domain contains90amino acids,classified toinitiator caspases.Three-dimensional structural model indicate that AjCASP1with the typicalfamily structure of α helices and β sheets,Multiple alignment and phylogenic analysis showedthat AjCASP1and the deduced CASP1of sea urchin were closely clustered in one group withthe highest similiarity.Blast analysis that the prodomain is CARD-CASP2in the preliminarydetermination to caspase-2,but there is a large different in dimensional structure with thehuman CASP2.Therefore,the subcellular localization and the mechanism in the signalingpathway maybe the following work.It was found that the Ajcasp1gene encoding amino acids with molecular weight of43kDa,and theoretical isoelectric point of5.82.A recombinat plasmid pGS21a-Ajcasp1containing the coding sequence of Ajcasp1gene was constructed using pGS-21a as a fusedexpression vector.The SDS-PAGE and Western blot revealed that the recombined protein wasexpressed successfully in Escherichia coli Rosetta(DE3) induced by IPTG.Then arecombinant fusion protein about60kDa was purified by Ni2+-NTA His-bind columnchromatography,and the polyclonal antibody was prepared.In order to further understand the fuction of AjCASP1,Western blot and Quantitativereal-time PCR revealed that AjCASP1in various tissues of sea cucumber.indicating that theprotein complex and widely distributed its assiociated structures in theorganization.Quantitative real-time PCR revealed that Ajcasp1mRNA expression levelsignificantly changed after LPS-induced, with the maximum expression24h followed by alower expression. The results give a reference for further study of the role of AjCASP1in thedevelopment and anti-bacterial infection in sea cucumber.It will helpful for understanding ofthe apoptosis mechinsim,as well as ontogenetic,morphogenetic and innate immune responseafter LPS stimulation provides an important theoretical reference.