文章摘要
郑清梅.斑节对虾溶菌酶基因的原核表达与产物活性检测[J].水产学报,2005,29(1):
斑节对虾溶菌酶基因的原核表达与产物活性检测
Expression of Penaeus monodon lysozyme gene in prokaryocyte system and evaluation of its lytic activity
投稿时间:2014-03-13  修订日期:2014-03-13
DOI:
中文关键词: 斑节对虾 溶菌酶基因 原核表达 溶菌活性
英文关键词: Penaeus monodon  lysozyme gene  prokaryocyte expression  lytic activities
基金项目:广东省自然科学基金
作者单位地址
郑清梅 中国水产科学研究院珠江水产研究所 中国水产科学研究院珠江水产研究所
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中文摘要:
      采用PCR方法对所克隆的斑节对虾溶菌酶基因的cDNA进行改造,并克隆至含温控启动子PRPL的大肠杆菌表达质粒pBV220,构建斑节对虾溶菌酶表达载体pBV220-lyz.该质粒转化大肠杆菌后经42℃诱导表达.SDS-PAGE电泳表明,表达产物中有16kD左右的特异性条带,与预期斑节对虾溶菌酶分子量相符.薄层扫描显示,重组斑节对虾溶菌酶约占全菌总蛋白的32%,纯化后的重组斑节对虾溶菌酶占全菌可溶性蛋白的90%左右.比浊法检测复性后产物的生物活性,结果表明重组斑节对虾溶菌酶的最适pH为6.0,最适温度为40℃.测定了重组斑节对虾溶菌酶对溶壁微球菌和几种鱼病原菌菌株的溶菌活力,结果表明,重组斑节对虾溶菌酶对溶壁微球菌、溶藻弧菌和鳗弧菌有较强的溶菌活力,对爱德华氏菌有一定的溶菌活力,对嗜水气单胞菌、副溶血弧菌和大肠杆菌DH5α溶菌活力较弱.
英文摘要:
      The c2type lysozyme cDNA of Penaeus monodon cloned previously in our lab (GenBank accession no. AF539466) was modified by PCR to delete the signal peptide and subcloned to pBV220 expression vector which contains PRPL promoter to construct the recombinant vector pBV2202lyz. pBV2202lyz was transformed to E. coli DH5αand then induced to express at 42 ℃. A specific band about 16kD in molecular mass was showed by SDS2PAGE analysis , which was larger than those from other species such as human , fish and insects (about 14kD) . The recombinant P. monodon lysozyme existed as inclusion bodies in the cells. The inclusion bodies were collected , washed and then dissolved. And finally the target protein was dialyzed in order to recover its biological activity. Scanned and quantified by the Analysis System of Biology Image showed that the recombinant lysozyme accounted for 32 % of the total cell protein , and was about 90 % after purification. The biological activity of the recombinant protein was evaluated by turbidimetric assay described by Hultmark. The optimum pH and temperature of the lytic activities of the recombinant lysozyme were 6.0 and40 ℃,respectively. The recombinant protein was found to possess potent lytic activities against Micrococcus lysodeikticus12634 , Vibrio alginolyticus As1. 1833 and V. anguillarum E3211. And it has some lytic activities against Edwardsiella tarda E 895205 but with little lytic activities against Aeromonas hydrophila AhG1 , V . parahaemolyticus As1. 1615 and E. coli DH5α. The standard lysozyme (lysozyme from chicken egg white) showed lower lytic activities against Micrococcus lysodeikticus12634 , Vibrio alginolyticus As1. 1833 and V. anguillarum E321 compared with that of the recombinant protein. The present study suggests the potential application of the recombinant P. monodon lysozyme in aqauculture.
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