Purification,Antibody Preparation And Application Of Cathepsin B From The Gut Of Sea Cucumber

Sea cucumber is an important aquatic animal and has been favored by consumers because of its abundant nutrition and bioactive substances.It is susceptible to change of several environmental factors,such as elevated temperature,UV exposure and high salt concentration.Autolysis of sea cucumber caused by these factors can lead to severe deterioration in its quality during handling and storage,and consequently cause heavy economic losses.Cathepsin was thought to be a major type of endogenous enzyme taking part in the autolysis of other marine animals.So its role in antolysis of sea cucumber deserves further study.In this study,purification and preparation of antibody of Cathepsin B from the gut of sea cucumber was investigated.And the distribution of it both in the body wall and gut of sea cucumber was preliminaryly explored.It was purified to homogeneity by ammonium sulfate precipitation,then chromatography with DEAE Sepharose CL-6B,Sephadex G-75 and TSK-GEL 3000 SWxl.The enzyme was purified 81.26-fold through the consecutive separation.The purified protease had one band on Native-PAGE and two protein bands with estimated Mw of 23 kDa and 26 kDa on SDS-PAGE.Z-Arg-Arg-MCA was a specific substrate.The enzyme displayed maximum activity at pH 5.5 and 45?.The enzyme activity appeared to be stable below 55 ?.The protease was scarcely inhibited by Ca2+and Mg2+,moderately inhibited by Mn2+,Fe2+and Fe3+and completely inhibited by Ni2+,Zn2+and Cu2+ions.Partial or complete inhibition was observed in the presence of thiol-blocking agents E-64 and indoacetic acid.Thiol-activating agents such as DTT,L-Cys and EDTA could enhance the activity.Serine-(PMSF,TI)and metallo-(1,10-phenanthroline)protease inhibitors partially inhibited the activity,but cysteine protease inhibitors,antipain and leupeptin evidently inhibited the activity of the purified protease.The results suggested that the purified protease belonged to cysteine protease and contained SH group(s).Purified enzyme was injected into a rat at multiple subcutaneous sites for the preparation of antibody.And the antibody was purified through affinity column on Protein G 2mL.The purity of the IgG was above 90%on SDS-PAGE.From the result of immunohistochemistry technique cathepsin B widely existed both in the body wall and gut of sea cucumber.