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| N-乙酰-D-神经氨酸醛缩酶基因的克隆与表达 | |
| 引用本文： | 杨蕴刘,饶娆,沈健,冯磊. N-乙酰-D-神经氨酸醛缩酶基因的克隆与表达[J]. 浙江大学学报(医学版), 2010, 39(1). DOI: 10.3785/j.issn.1008-9292.2010.01.010 |
| 作者姓名： | 杨蕴刘 饶娆 沈健 冯磊 |
| 作者单位： | 1. 中科院上海植物生理研究所,上海,200032 2. 浙江医学高等专科学校,浙江,杭州,310053 3. 浙江大学医学院,浙江林学院健康管理系,浙江,杭州,310058 |
| 摘 要： | 目的:为了获得N-乙酰-D-神经氨酸醛缩酶基因表达的两重组菌.方法:以大肠杆菌C600染色体DNA为模板,通过引物设计和PCR扩增,克隆获得编码Neu5Ac醛缩酶基因(nanA)的片段;以pET28b构建的nanA基因重组质粒pRY1转化 E.coli BL21(DE3),然后将nanA基因片段组入到pDR540载体的Ptac启动子下游,所得重组菌 E.coli DH5α/pRY3的nanA基因在tac启动子控制下呈组成型表达.结果:DNA序列测定证明,该基因的开放阅读框(open reading frame,ORF)大小为894 bp, 编码298个氨基酸组成的酶蛋白;SDS-PAGE显示,纯化的目的蛋白为33 kD的单一条带.以pET28b构建的nanA基因重组质粒pRY1转化 E.coli BL21(DE3),在得到的转化子 E.coli BL21(DE3)/pRY1中,nanA基因受lac启动子控制,无需IPTG或乳糖所诱导.在N-乙酰(-D)-神经氨酸醛缩酶固定化酶的催化下合成N-乙酰-D-神经氨酸的合成效率为78.3%,结晶回收率为90.2％,总回收率为70.6%.全波长扫描分析表明:本实验室结晶品与Sigma公司商品的全波长扫描图谱有相同的特征.结论:所构建的诱导型产酶菌株 E.coli BL21(DE3)/pRY1和组成型表达菌株 E.coli DH5α/pRY3都可较多量的产酶.合成的结晶品具有N-乙酰-D-神经氨酸的特征. |
| 关 键 词： | N-乙酰神经氨酸/遗传学 果糖-二磷酸醛缩酶 大肠杆菌/遗传学 聚合酶链反应 克隆 分子 重组 遗传 转染 遗传载体 基因表达 重组蛋白质类 |
Cloning and expression of N-Acetyl-D-neuraminic acid aldolase in Escherichia coli |
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| YANG Yun-liu,RAO Rao,SHEN Jian,FENG Lei. Cloning and expression of N-Acetyl-D-neuraminic acid aldolase in Escherichia coli[J]. Journal of Zhejiang University. Medical sciences, 2010, 39(1). DOI: 10.3785/j.issn.1008-9292.2010.01.010 | |
| Authors: | YANG Yun-liu RAO Rao SHEN Jian FENG Lei |
| Affiliation: | YANG Yun-liu 1,RAO Rao 1,SHEN Jian2,FENG Lei 3(1.Shanghai Institute of Plant Physiology,Chinese Academy of Sciences,Shanghai 200032,China,2.Zhejiang Medical College,Hangzhou 310053,3.College of Medicine,Zhejiang Forestry University,Zhejiang University,Hangzhou 310058,China) |
| Abstract: | Objective: To obtain the Escherichia coli strains expressing N-Acetyl-D-neuraminic acid aldolase(Neu5Ac aldolase).Methods: The gene(nanA) coding Neu5Ac aldolase was cloned from Escherichia coli C600,and the recombinant plasmid was sequenced and expressed in Escherichia coli.Results: Sequencing data revealed that the open reading frame was 894 bp and predicted to encode a protein consisting of 298 amino acids.The patterns of SDS-PAGE showed that the purified enzyme protein as a single protein band with a mol... |
| Keywords: | N-Acetylneuraminic acid/genet Fructose-bisphosphate aldolase Escherichia coli/genet Polymerase chain reaction Cloning,molecular Recombination,genetic Transfection Genetic vectors Gene expression Recombinant proteins |
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