Lectin From Manila Clam (Ruditapes Philippinarum) And Its Inhibitory Mechanism Against Shewanella Putrefaciens

MCL(Manila clam Lectins)is one of the important humoral factors in the immune system of Ruditapes philippinarum,which inhibits the growth of many microorganisms.When Manila clam Lectins is infected by external microorganisms,the amount of MCL will increase dramatically in the body of clams.In this study,S.putrefaciens was used to infect Manila clam to stimulate the body to produce MCL,and then the biological activity,antibacterial activity,antibacterial mechanism and preservation effect of MCL were explored in order to provide basic data to follow up research and application of MCL.The main contents and results of this paper are as follows:1 Determined the temporal pattern of MCL appeared in Manila clam by measuring hemagglutination activity of haemolymph from Manila clam in different months;Different treatment methods of SPS injected(S.putrefaciens suspension),stab wounds and SPS soaking were used to explore the optimal inducing source for stimulating MCL production;Different ammonium sulfate salting methods were used to optimize the MCL extraction process.The results show that Manila clam contained less MCL in the hemolymph under normal conditions.The Ushaped 96-well plate method could not detect the hemagglutination activity in the hemolymph;injection of SPS into the Manila clam could stimulate the secretion of Manila clam.The stimulatory effect of SPS injected is better than stab wound,SPS immersion two ways.When SPS reaches an optical density of 0.5 at a wavelength of 600 nm,it is the best concentration for stimulating the secretion of MCL from the Manila clam.The pH of the mixture which added ammonium aimed to salting out MCL should be control to around 7.0.2 The MIC value of MCL to S.putrefaciens was determined by broth dilution method;the effect of MCL on the growth curve of S.putrefaciens was measured by spectrophotometry;The effect of MCL on membrane and the cell wall integrity of S.putrefaciens was detected by measuring the activity of AKP enzyme and OD260 in the culture medium.;Ultrastructure of S.putrefaciens was observed by scanning electron microscopy.The results show that the MIC value of MCL on S.putrefaciens could not dectect in the range from 0.024 mg/mL to 12.5 mg/mL.MCL could significantly delay the arrival of the logarithmic growth phase of S.putrefaciens when its concentration was between 0.00024 mg/mL and 1 mg/mL.When S.putrefaciens is in logarithmic growth phase,the inhibition of growth of S.putrefaciens by MCL is negatively correlated with the concentration of MCL;when S.putrefaciens is in a stable growth phase,the inhibitory effect of MCL on its growth and the concentration of MCL.Positive correlation.MCL can destroy cell walls and cell membranes of cells.High concentrations of MCL co-culture with S.putrefaciens can agglutinate the bacterias and form a layer of membrane-enveloping bacteria around the cell walls.3 Using unlabeled quantitative proteomics method,we analyzed the protein expression profile of S.putrefaciens cells after 6 h treatment with MCL(L group)and normal S.putrefaciens(C group).A total of 421 proteins were identified,of which 74 were differential proteins.The expression of 16 proteins was up-regulated and the expression of 58 proteins was down-regulated.Analysis of proteomics showed that the expression levels of pyrophosphokinase and enolase(AC:A1S4D7),the phosphorylase ribozymes involved in the amino acid biosynthetic pathway,were up-regulated,while the expression levels of the remaining 10 proteins were down-regulated;the pathway involved in pyruvate metabolism,citric acid/ The protein expression levels of the tricarboxylic acid cycle pathway,aminoacyl-tRNA biosynthesis pathway,and carbon metabolism pathway were all down-regulated.This shows that MCL inhibits the bacteria by blocking amino acid synthesis in S.putrefaciens,inhibiting pyruvate metabolism,and citrate circulation.4 Fresh catfishes were used as experimental objects to investigate the preservation effect of MCL with the total number of colonies,TVB-N value,K value,malondialdehyde value,and pH value as the freshness indicators.The results showed that MCL could reduce the total number of colonies,TVB-N value,K value and malondialdehyde value during the fresh-keeping process of carp,and had no significant effect on the pH value of catfishes.This shows that MCL has a certain effect on the preservation of fish.