Study On The Biological Activities Of Catesbeianalectin And Screening Its Saccharide Ligand

Bioactive peptide is a kind of peptide with physiological action,which is good for the life activity of organism.Bioactive peptide has much function,such as antimicrobial,anticancer,antivirval,et al.At present,people have paid more attention to bioactive peptide,duing to its advantage.A kind of bioactive peptide that could agglutinate erythrocyte is referred to as lectin.Lectin is a kind of protein which could combine with giycoprotein or sugar specially.Lectin was the potencial candidate for drug delivery and targeting,but the molecular weight of the natural lectin was big,causing its toxicity and immunogenicity.The molecular weight of lectin which was known at home and abroad,was more than 10 KDa.In this study,Catesbeianalectin was the smallest lectin with high agglutinating activity so far,which was obtained by the isolation and identification of the skin secretion of Rana catesbiana,and the construction of cDNA library of Bullfrog skin,its molecular weight was just 1465.8 Da.In order to research the structural characteristics,bioactivity and its special saccharide ligand,Catesbieanalectin was synthetized,its secondary structure was determined and analyzed by FT-IR and CD.The spectra of FT-IR showed that the strong peak in 1669.54 cm^-1was the characteristic absorption peak of random coil,the band between 1600 and 1700 cm^-1and the band between 1200 and 1330 cm^-1were respectively the characteristic band of alpha helix and beta folding.Deconvoluted acid 1 band and Gaussian curve-fitting was obtained via FT-IR and computer,the content of various structures was gained by calculating peak height and peak area,alpha helix accounted for 10%,beta folding accounted for 40%,beta turn accounted for 20%,and random coil accounted for 20%.The secondary structure of Catesbeianalectin could not accurately comfirmed because of the overlap among characteristic peaks.Thus,the secondary structure of Catesbeianalectin was further determined by CD,the CD spectra showed there was a characteristic absorption peak in21-225nm,where was the peak of structure of PPII.In order to investigate the bioactivities of Catesbeianalectin,hemagglutination test and bacterial agglutination test about Catesbeianalectin were carried out.The results showed the MIC of Catesbeianalectin to erythrocyte was 10?g/mL,and the MIC to Staphylococcus aureus,streptococcus,APP,salmonella were respectively 2.5?g/mL?1.25?g/mL?5?g/mL?5?g/mL.After that,the immunogenicity of Catesbeianalectin was also detected.The Catesbeianalectin,OVA,WGA were prepared to oil emulsion antigen,which was used to vaccinate Balb/c mouse that was 5-6 weeks old.The immunogenicity of Catesbeianalectin was detected and analized by routine blood test and indirect ELISA.The result of routine blood test showed Catesbeianalectin had effects on the number of neutrophils,lymphocyte rate and the number of soterocyte.The result of indirect ELISA showed that the titer of serum of test group 1 reached 1:200,when the coating concertration of OVA was 0.25?g/mL;the titer of serum of test group 2 reached 1:1600,when the coating concertration of WGA was 0.0078?g/m L;the titer of serum of test group 3 reached 1:25,when the coating concertration of Catesbeianalectin was 80?g/mL.The results indicated that Catesbeianalectin had low or none immunogenicity.The interaction between Catesbeianalectin and sugar was surveyed by SPRi,the association and dissociation were real time monitored.The result showed there was no obvious interaction between Catesbeianalectin and sugar for test,when 0.4 mg/mL Catesbeianalectin passed though the 30 sugars which fixed on the biochip.