title: Gene identification and recombinant protein of a lysozyme from freshwater mussel Cristaria plicata.
abstract: Lysozymes are important proteins to bivalve in the innate immune responses against bacterial infections, and provide nutrition as digestion enzymes. A new LYZ1 from the freshwater mussel Cristaria plicata was cloned by rapid amplification of cDNA ends (RACE) and nested PCR method. The full-length cDNA sequence of CpLYZ1 was 763 bp. The cDNA contained a 5'-terminal untranslated region (UTR) of 21 bp, a 3'- terminal UTR of 259 bp with a 29 bp poly(A) tail, a tailing signal (AATAAA) and the open reading frame of 483 bp. The CpLYZ1 cDNA encoded a polypeptide of 160 amino acids with a predicted molecular mass of 17.8 kDa, and a theoretical isoelectric point of 6.07. The comparison of the deduced amino acid sequences with LYZs from other species showed that the enzyme belonged to i-type lysozyme. The mRNA transcript of CpLYZ1 could be detected in all the examined tissues with the highest expression level in hepatopancreas. The expression levels of CpLYZ1 in hemocytes, hepatopancreas and gill significantly increased after Aeromonas hydrophila challenge. The expression level of CpLYZ1 in hemocytes sharply decreased from 6 h to 24 h and significantly increased at 48 h, and was the highest level in hepatopancreas at 24 h, and was the maximum level in gill at 48 h. Furthermore, the recombinant CpLYZ1 was induced to be expressed as an inclusion body form by IPTG at 37 °C for 4 h, and then was purified by using the Ni(2+) affinity chromatography. The relative enzyme activity of the recombinant CpLYZ1 was influenced on pH and temperature. The optimal pH and temperature was 5.5 and 50 °C, respectively. Against Escherichia coli, A. hydrophila, Staphyloccocus aureus, Bacillus subtilis, Streptococcus sp. and Staphylococcus epidermidis, the recombinant CpLYZ1 had bacteriolytic activity.
authors: Department of Bio-science, Institute of Life Science, Nanchang University, 999 Xuefu Road, Nanchang 330031, Jiangxi Province, China.
authors: Dan
authors: D
authors: Wu
authors: Baoqing
authors: B
authors: Hu
authors: Chungen
authors: C
authors: Wen
authors: Gang
authors: G
authors: Lin
authors: Zhiying
authors: Z
authors: Tao
authors: Xiaojuan
authors: X
authors: Hu
authors: Yanhai
authors: Y
authors: Xie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006090:Gram-Negative Bacteria / Q000502:physiology; D006094:Gram-Positive Bacteria / Q000502:physiology; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D009928:Organ Specificity; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D049876:Unionidae / Q000737:chemistry / Q000201:enzymology / Q000235:genetics* / Q000276:immunology*
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D009113:Muramidase