Study On The Expression,purification And Function Of Apostichopus Japonicus Catalase

Sea cucumber is an important economic aquaculture species in the coastal areas of China.In recent years,intensification led to the deterioration of its breeding environment and frequent disease problems.Therefore,it is important to understand the factors that influence and regulate the immune of sea cucumber.As an important antioxidant enzyme,catalase（CAT）widely distributed in many tissues and removed the excessive H₂O₂.Meanwhile,it shows that the catalase also plays a role in the intestinal immune of biological organisms.We detected the expression of the Apostichopus japonicus catalase（Aj-CAT）gene by qRT-PCR.The results showed that Aj-CAT had corresponding expression in all tissues,and had high expression level in the body wall and antennae.The Apostichopus japonicus were challenged with pathogenic bacteria via oral infection,the expression level of Aj-CAT at the intestine was upregulated,and the enzyme activity was increased in the intestine.These results suggested that Aj-CAT have a important role for Apostichopus japonicus intestinal immunityThe full length of Aj-CAT encoded by 1503 base pairs,and translated into 500amino acids analyzed by the bioinformatics methods.The online software ExPASy compute its pI/Mw,.It demonstrated thatthe theoretical isoelectric point pI of Aj-CAT is 6.54,and the molecular weight of the protein is 56.56 kDa.The phylogenetic tree was constructed by homology comparison with the MEGA 7.0.It showed that Aj-CAT protein was closely related to the Perinereis aibuhitensis catalase.Then we expressed the Aj-CAT protein in vitro.t.First,the Aj-CAT gene with Nco I/Xho I restriction site and His-tag was cloned using the plasmid stated in our laboratory.We construted the clone plasmid pMD18-His-Aj-CAT（Nco I/Xho I）.The pMD18-His-Aj-CAT and expression vector pET28c were double-digested,and the recombinant expression vector pET28c-His-Aj-CAT and its expression host pET28c-His-Aj-CAT/BL21（DE3）were constructed.The H₂O₂ biotolerance experiment was performed on recombinant genetically engineered bacteria after induced by IPTG.The results of SDS-PAGE,Western blot and the enzyme activity demonstrated that the host could express the activity rAj-CAT protein.The crude enzyme solution containing rAj-CAT was successfully obtained by Ni²⁺-NTA affinity chromatography This study laid a foundation for further research,development and application of catalase from sea cucumber.