Expression Analysis Of I-type Lysozyme Gene From Cristaria Plicata And Enzymatic Activity Analysis

Two i-type lysozymes (CpLYZ1,CpLYZ2) cDNAs were cloned from freshwater mussle, Cristaria plicata, using rapid amplification of cDNA ends and nested PCR.The full-length cDNA sequence of CpLYZ1was763bp (GenBank accession number:JQ988052), consisting of a5’-terminal untranslated region (UTR) of21bp, a3’-terminal UTR of259bp with a29bp poly (A) tail, a tailing signal (AATAAA) and a ATTTA (RNA instability motifs), and the open reading frame of483bp. The CpLYZl cDNA encoded a polypeptide of160amino acids with a predicted molecular mass of17.8kDa, and a theoretical isoelectric point of6.07. The N-terminus had the features consistent with a signal peptide as defined by SignalP software with a putative cleavage site located after position23(MEAVKMFFFLVGLLLTFNKCA ES), the deduced mature peptide was of137amino acid residues, and the mature peptide harbored fourteen cysteine residues. The deduced amino sequence of CpLYZl contains two highly conserved i-type lysozyme activity sites (Glu61and Ser72); i-type specific motif:C-L-[E/L/R/H]-C-[I/M]-C; and the conserved motif MDVGSLSCG(P/Y)(Y/F)QIK in bivalve i-type lysozymes.The full-length cDNA sequence of CpLYZ2was913bp (GenBank accession number:JQ988051), consisting of a5’-terminal untranslated region (UTR) of38bp, a3’-terminal UTR of389bp with a29bp poly (A) tail, a tailing signal (AATAAA) and a ATTTA (RNA instability motifs), and the open reading frame of486bp.The CpLYZ2cDNA encoded a polypeptide of161amino acids with a predicted molecular mass of18.2kDa, and a theoretical isoelectric point of6.56. The N-terminus had the features consistent with a signal peptide as defined by SignalP software with a putative cleavage site located after position23(MEAVKMFFFLVGVLLTFTKCTES), the deduced mature peptide was of138amino acid residues, and the mature peptide harbored twelve cysteine residues. The deduced amino sequence of CpLYZ2contains only one highly conserved i-type lysozyme activity sites (Glu61).It has i-type specific motif:C-L-[E/L/R/H]-C-[I/M]-C, and the part of the conserved sequence of amino acids in bivalve i-type lysozymes. The expression of CpLYZ41mRNA and CpLYZ2mRNA in Cristaria plicata were measured by fluorescent real-time quantitative RT-PCR. The results showed that the mRNA expression of CpLYZ1and CpLYZ2had significant differences in the organization. The expression level of CpLYZl in hepatopancreas was the highest. It is5.23times higher than that of hemocytes. Followed by the gill, it was1.67times higher than that of hemocytes. The expression level of CpLYZ1in muscle and mantle are the lowest. They were both lower than that of hemocytes, and were0.18times and0.32times of hemocytes respectively. The expression level of CpLYZ2in gill was the highest. It was3.85times higher than that of hemocytes.The expression levels of CpLYZ2in hepatopancreas, muscle, mantle and hemocytes had no significant differences.Fluorescent real-time quantitative RT-PCR showed the expression levels of CpLYZ1and CpLYZ2in hemocytes, hepatopancreas and gill were increased significantly after the pathogenic bacteria Aeromonas hydrophila challenged. The maximum levels of CpLYZ1and CpLYZ2in hemocytes were both at48h. They were respectively7.16times and4.12times higher than blank control. The maximum levels of CpLYZ1and CpLYZ2in hepatopancreas were both at24h. They were respectively34.46times and8.64times higher than blank control. The maximum level of CpLYZl in gill was at48h, it was2.73times higher than blank control. The maximum level of CpLYZ2in gill was at12h, it was18.44times higher than blank control.The expression levels of CpLYZl and CpLYZ2in hemocytes, hepatopancreas and gill have some changes after PBS challenged. In hemocytes, the expression levels of CpLYZ1at6,12and24h were all lower blank control. The maximum level was at48h, it was7.16times higher than blank control. The expression level of CpLYZ2was first increased, then decreased and then increased. The maximum level was at48h, it was2.35times higher than blank control. In hepatopancreas, the expression level of CpLYZl was first decreased, and increased. The maximum level was at48h, it was6.16times higher than blank control.The expression level of CpLYZ2was the hihhest at48h, which was1.68times higher than t blank control. In gill, the expression level of CpLYZ1downward trend, it was0.27times of blank control. The expression level of CpLYZ2slightly increased, the level at48h was2.48times of blank control.In order to further inquire into the functions of CpLYZl and CpLYZ2, pET30-LYZ1and pET30-LYZ2prokaryotic expression system were constructed by double digestion, and prokaryotic expression plasmid was transformed into E.coli (DE3). Two recombinant proteins were successfully expressed after IPTG induction. Two recombinant proteins are not soluble form of inclusion bodies proteins. The inclusion bodies were dissolved by the denaturant, were purified by using the native Ni2+affinity chromatography, and refolding of them using the method of gradient dilution.The relative enzyme activity of two recombinant proteins examined found that the recombinant protein existing as the inclusion body was not biologically active. Howerve, the recombinant CpLYZ protein has obvious lysozyme activity after denaturation and renaturation. Micrococcus lysodeikticus as substrate, the optimum pH of the recombinant CpLYZl and CpLYZ2was5.5, and the optimum temperature was50℃. Against E.coli, Aeromonas hydrophila, Staphyloccocus aureus, Bacillus subtili, Streptococcus and Staph Epidermidis, the recombinant CpLYZ has bacteriolytic activity. Their bacteriolytic activities were both the strongest against Bacillus subtili. At the same time, they have the inhibitory function against two gram-negative bacteria. The bacteriolytic activity of CpLYZl against six bacteria is higher than CpLYZ2. The recombinant CpLYZl and CpLYZ2still had bacteriolytic activity against Micrococcus lysodeikticus after they are treated by100℃for50min, but the activity was greatly reduced.Rabbit polyclonal antibody was prepared using purified recombinant CpLYZ2protein. The serum antibody titers measured by ELISA was1:512000, and high titer rabbit polyclonal antibody was gained. Western blotting showed that the recombinant CpLYZ2protein was able to combine with rabbit polyclonal antibody specifically. At the same time, the expression of CpLYZ2was detected in the hemocytes of Cristaria plicata.