磷酸丝氨酸转氨酶(serC)基因的克隆与原核表达

目的]检测磷酸丝氨酸转氨酶(serC)基因在大肠杆菌中能否高效表达.方法]根据GenBank所公布的serC基因序列设计一对特异性引物,以E.coliJM109基因组为模板PCR扩增目的基因片段,将得到的目的基因定向克隆至大肠杆菌/黄色短杆菌穿梭表达载体pEC7中,构建重组表达载体并转化宿主菌E.coli BL21(DE3),经IPTG诱导表达后进行SDS-PAGE分析.结果]通过PCR扩增得到约1 100 bp的DNA片段,经测序后分析该基因与已发表的serC基因具有99.27;的同源性;对重组表达载体进行酶切和PCR方法鉴定正确的命名为pEC7- C;重组表达载体转化宿主菌,SDS-PAGE分析可见约41 kD与理论大小一致的目标蛋白条带.结论]重组表达载体转化后E.coli BL21中蛋白表达量比原始菌株的蛋白表达量高,证明 serC基因在E.coli BL21(DE3)中成功表达,为进一步构建L-丝氨酸高产基因工程菌奠定了基础.

Cloning and Prokaryotic Expression of Phosphoserine Transaminase (serC) Gene

【Objective】To test whether the serC gene in E.coli efficiently expressed.【Method】Based on DNA sequence encoding phosphoserine transaminase(serC) reported on the GenBank,serC gene was cloned from E.coli JM109 by PCR method,and the serC gene was cloned into pEC7 which is a Escherichia coli/Brevibacterium flavum shuttle expression vector.The recombinant plasmid pEC7-C was transformed into Escheriehia coli BL21,and the positive clone was induced with IPTG,another expression were analyzed by SDS-polyacrylamide gel electrophoresis.【Result】The PCR product was approximately 1 100 bp.Sequencing analysis showed that it shared more than 99% homology with the corresponding sequences published.Subsequently,SDS-po-lyacrylamide gel electrophoresis indicating that about 41 kD protein was obtained.【Conclusion】After the transformation of recombination expression vehicle,the expression volume in E.coli BL21(DE3) was higher than that in original strain,which verified successful expression of serC gene performed in E.coli BL21(DE3),which set the base for further construction L-serine genetic engineering Brevibacterium flavum.