The Analysis Of The Immunologic Function,Isopeptidase Activity And Detection Of The Key Sites Of The Antibacterial Activity For The Stichopus Japonicus Lysozyme

Sea cucumber is an important economic breeding species in coastal cities of China.In recent years,the cultured environment and the disease are becoming more and more serious.Studies have shown that in the face of microbial infection,sea cucumber own two immune stress strategies.The one way was antimicrobial peptides,such as lysozyme,usually directly destroy the pathogenic bacteria;the another way was to produce excess reactive oxygen species(Reactive oxygen species,ROS).However,excessive ROS was a serious toxic effect.But the antioxidant defense system(SOD and CAT)of the organism can balance the ROS and maintain the redox balance of the body.Therefore,it is important to study the related immune mechanism of sea cucumber,and to understand the nature and function of immune-related proteins will be able to effectively enhance the resistance of sea cucumber to play an important role in the further study of antioxidant and immune function.In this study,the effects and functions of lysozyme and catalase were discussed.The distribution of sea cucumber i-type lysozyme in five tissues was analyzed for the first time,and the immune function of sea cucumber lysozyme was preliminarily determined.The results showed that the amount of sea cucumber lysozyme in the tentacles was the most,followed by the respiratory tree,and in the intestine at least.The coelomocytes and body wall were basically flat.The content of lysozyme in sea cucumber was increased at 48 h,and then the content of lysozyme in sea cucumber was decreased gradually.At the cellular level,the immunosuppressive effects of sea cucumber i type lysozyme on pathogens were analyzed by gene silencing technique.After gene silencing for lysozyme,the expression of lysozyme protein in the body cavity cells was inhibited by about 50%at the transcriptional level,and the apoptosis and necrosis of the cultured cells were significantly increased under the same immunological conditions.In vitro,the active site of the sea cucumber i-type bacteriocin was determined by the site-directed mutagenesis technique.The Trx-His-S tag peptide of recombinant lysozyme was excised to obtain the non-fusion lysozyme,and the antibacterial activity and isomeric peptidase activity of lysozyme were analyzed.In the experiment,the recombinant Escherichia coli intracellular expression gene engineering bacteria of sea cucumber catalase gene sequence was constructed,and the target protein was successfully expressed,and then optimize its expression conditions.The recombinant plasmid pET28c(+)-CAT/BL21(DE3)was successfully constructed.We then optimized the expression conditions,such as time,IPTG,temperature,pH,speed.The optimal expression conditions were as follows:the amount of IPTG was 0.05 mmol/L,pH 7.4,25?,180 rpm,and the induction time was 6 h.This study provides a good theoretical basis for the deep research,development and utilization of sea cucumber lysozyme,and provides a theoretical basis for the future separation and purification of sea cucumber catalase.