大肠杆菌植酸酶appA2基因真核表达载体的构建及在细胞中的表达

Construction of eukaryotic expression vector and expression of appA2 gene of Escherichia coli in PK15

根据已经公布的植酸酶appA2基因序列,设计合成了1对特异性引物,应用RT-PCR技术从大肠杆菌中扩增得到植酸酶appA2基因,并将其克隆到真核表达载体pcDNA3.1(+)中,构建了重组真核表达质粒pcDNA-appA2,对重组表达质粒鉴定正确后,转染猪PK15细胞,经G418筛选后,通过实时荧光定量PCR检测细胞内appA2表达,同时测定细胞内植酸酶的活性,检测结果表明,本试验成功构建了pcDNA-appA2重组真核表达载体,转染细胞后appA2的表达量是对照组的1 686.55倍,同时具有较好的植酸酶活性,为通过生物反应器制备植酸酶研究奠定了基础。

According to the published sequence of Phytase appA2 gene,a pair of primers were designed and synthesized.Phytase appA2 gene was amplified by RT-PCR from Escherichia coli,and cloned into pcDNA3.1（＋） vector.The eukaryotic expression vector pcDNA-appA2 was successfully constructed.Phytase appA2 gene was sequenced and compared with the published sequence of Phytase appA2 gene in GenBank.The expression of recombinant plasmid pcDNA-appA2 in PK15 cells was induced and detected by Quantitative fluorogenic real-time PCR assay for appA2 mRNA expression level and modified Molybdate colorimetric method for enzyme activity.The results showed that the recombinant vector pcDNA-appA2 was successfully constructed.After transfection,Phytase expression level in pcDNA-appA2 cell group was 1686.55 times greater than that of control and it has good enzymatic activities.