摘要
目的应用重叠PCR技术,构建节杆菌DMDC12的Mn-SOD基因sodAP的重组表达质粒,并在E.coliBL21(DE3)中表达。方法设计两对引物,分别扩增启动子P121和sodAP基因序列,利用重叠PCR技术,将启动子P121与sodAP基因相连接并测序,连接产物插入原核表达质粒pET-22b(+),构建重组质粒pET-PsodAP,转化E.coliBL21(DE3)进行表达。用SDS-PAGE检测表达产物,光密度定量分析法分析目的蛋白表达量。结果启动子P121和sodAP基因的连接产物经凝胶电泳检测,可见约800bp的目的条带,测序结果与预期相符;重组表达质粒pET-PsodAP经双酶切鉴定,表明构建正确;目的蛋白在E.coliBL21(DE3)中获得了表达,表达量占菌体总蛋白的19%。结论应用重叠PCR技术,成功构建了sodAP基因的重组表达质粒,并在E.coliBL21(DE3)中获得表达,为Mn-SOD的进一步工业化生产奠定了基础。
Objective To construct an recombinant plasmid for expression of sodAP gene ofArthrobacterpascens DMDC12 in E. coil BL21 （DE3） by over lap PCR. Methods Promoter P121 and sodAP gene were amplified using two pairs of designed primers respectively, then linked by over lap PCR, identified by sequencing and inserted into prokaryotic expression vector pET-22b （＋）. Transform the constructed recombinant plasmid pET-PsodAP to E. coli BL21 （DE3） and identify the expressed product by SDS-PAGE. Determine the expression level of target protein by quantitative photodensitometry. Results The linked PI21 promoter and sodAP gene showed a band of about 800 bp on gel electrophoretic profile, and its sequence was consistent with that respected. Restriction analysis proved that recombinant plasmid pET-PsodAP was constructed correctly. The expressed product contained 19% of total somatic protein. Conclusion The recombinant expression vector for sodAP gene was successfully constructed by over lap PCR technique and expressed in E. coli BL21（DE3）, which laid a foundation of industrial production of Mn-SOD.
出处
《中国生物制品学杂志》
CAS
CSCD
2008年第9期762-764,共3页
Chinese Journal of Biologicals
