【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.coliBL21(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.coliDH5α/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 read ing 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.coliBL21(DE3),在得到的转化子E.coliBL21(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.coliBL21(DE3)/pRY1和组成型表达菌株E.coliDH5α/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。更多还原

【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 molecular weight of 33 kD,which was consistent with those reported in the reference.In the recombinant plasmid pRY1,the expression of nanA gene was controlled by the lac promoter with the induction of IPTG or lactose.The plasmid pRY3 was constructed,in which the nanA gene ws controlled by the tac promoter.The protein of Neu5Ac aldolase was constitutively expressed using the recombinant strain,E.coli DH5α/pRY3 without induction of IPTG or lactose.The crystal was finally obtained with the efficiency of 90.2% of Neu5Ac.The HPLC indicated that the Neu5Ac crystal prepared in this experiment was same as Simga product.Conclusion: The protein products expressed by two recombinant strains E.coli BL21(DE3)/pRY1 and DH5α/pRY3 has the characteristics of Neu5Ac.更多还原