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论文类型：期刊论文

发表时间：2011-05-01

发表刊物：PROTEIN EXPRESSION AND PURIFICATION

收录刊物：SCIE、PubMed

卷号：77

期号：1

页面范围：20-25

ISSN号：1046-5928

关键字：Apostichopus japonicus; Pichia pastoris; Lysozyme; Expression; Antimicrobial activity

摘要：Apostichopus japonicus (sea cucumber) is one of the economically important farmed echinoderm species in Northern China. As a crucial enzyme in innate immunity, lysozyme plays a key role in the overall defense against pathogens in A. japonicus. In the present study, a lysozyme gene from A. japonicus was cloned by PCR and expressed in Pichia past oris using the expression vector pPIC9K. The expressed lysozyme had a molecular mass of similar to 14 kD, as shown by SDS-PAGE and Western-blotting. The expression condition was optimized, and the highest expression level was achieved by induction with 1% methanol at pH 5.0 for 120 h. The recombinant lysozyme was purified by affinity chromatography using a Ni-NTA column. The specific activity of the purified lysozyme was 34,000 U/mg using Micrococcus lysodeikticus as substrates. It exhibited antimicrobial activity toward M. lysodeikticus, as detected by growth inhibition on agar plate and turbidity assay, suggesting a potential application of A. japonicus lysozyme as an antimicrobial agent in A. japonicus aquaculture. (c) 2011 Elsevier Inc. All rights reserved.