Molecular cloning of an invertebrate goose-type lysozyme gene from Chlamys farreri, and lytic activity of the recombinant protein.
Molecular cloning of an invertebrate goose-type lysozyme gene from Chlamys farreri, and lytic activity of the recombinant protein.
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DOI：
10.1016/j.molimm.2006.06.008
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发表时间：
2007-02
影响因子：
3.6
通讯作者：
Jianmin Zhao;L. Song;Chenghua Li;Huibin Zou;Duojiao Ni;Wan Wang;W. Xu
中科院分区：
文献类型：
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作者：
Jianmin Zhao;L. Song;Chenghua Li;Huibin Zou;Duojiao Ni;Wan Wang;W. Xu
溶菌酶是一种广泛分布的水解酶，具有裂解细菌肽聚糖的活性，使其能够保护宿主免受病原体感染。采用表达序列标签（EST）和cDNA末端快速扩增（RACE）技术，从栉孔扇贝（Chlamys farreri）中克隆了一种无脊椎动物鹅型溶菌酶（CFLysG）的cDNA。CFLysG基因全长829个核苷酸，含有一个典型的多聚腺苷酸化信号序列AATAAA和一个poly（A）尾，开放阅读框（ORF）为603 bp，编码200个氨基酸残基的多肽，预测分子量为21.92 kDa，理论等电点为7.76。CFLysG与脊椎动物中鹅型（g型）溶菌酶的高度相似性表明CFLysG应该是g型溶菌酶家族的无脊椎对应物，这表明g型溶菌酶的起源早于尾索动物的出现，甚至早于后口动物的出现。CFLysG与大多数G型溶菌酶相似，具有G型溶菌酶的基本结构和功能所必需的所有保守特征，如3个催化残基（Glu 82，Asp 97，Asp 108）。北方杂交结果表明，CFLysG基因在鳃、肝胰腺和性腺组织中表达量最高，在血细胞和外套膜组织中表达较弱，在闭壳肌中未检测到。这些结果表明，CFLysG可能具有免疫和消化适应性溶菌酶的综合特性。为了深入了解CFLysG的体外裂解活性，将成熟肽编码区克隆到毕赤酵母中进行异源表达。重组CFLysG对革兰氏阳性菌和革兰氏阴性菌均有抑制作用，且对革兰氏阳性菌的抑制作用更强，表明CFLysG参与了C.法勒里
Lysozyme is a widely distributed hydrolase possessing lytic activity against bacterial peptidoglycan, which enables it to protect the host against pathogenic infection. In the present study, the cDNA of an invertebrate goose-type lysozyme (designated CFLysG) was cloned from Zhikong scallop Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of CFLysG consisted of 829 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 603bp encoding a polypeptide of 200 amino acid residues with a predicted molecular weight of 21.92kDa and theoretical isoelectric point of 7.76. The high similarity of CFLysG with goose-type (g-type) lysozymes in vertebrate indicated that CFLysG should be an invertebrate counterpart of g-type lysozyme family, which suggested that the origin of g-type lysozyme preceded the emergence of urochordates and even preceded the emergence of deuterostomes. Similar to most g-type lysozymes, CFLysG possessed all conserved features critical for the fundamental structure and function of g-type lysozymes, such as three catalytic residues (Glu 82, Asp 97, Asp 108). By Northern blot analysis, mRNA transcript of CFLysG was found to be most abundantly expressed in the tissues of gills, hepatopancreas and gonad, weakly expressed in the tissues of haemocytes and mantle, while undetectable in the adductor muscle. These results suggested that CFLysG could possess combined features of both the immune and digestive adaptive lysozymes. To gain insight into the in vitro lytic activities of CFLysG, the mature peptide coding region was cloned into Pichia pastoris for heterogeneous expression. Recombinant CFLysG showed inhibitive effect on the growth of both Gram-positive and Gram-negative bacteria with more potent activities against Gram-positive bacteria, which indicated the involvement of CFLysG in the innate immunity of C. farreri.