pEGFP-植酸酶基因质粒重组构建及其在COS7细胞中表达分析

Construction of Bacterial Phytase Gene in pEGFP-N_3 Vectors and Its Expression in COS7 Cells

采用PCR技术扩增细菌酸性植酸aPPA2基因ORF序列,其DNA分子为1 299 bp,编码432氨基酸,蛋白质分子量约为48 kD a。此植酸酶基因被克隆到pEGFP-N3表达载体的BamH1和Pst1克隆区域,重组的pEG-FP-aPPA2重组质粒经转化到哺乳类培养细胞COS7中。重组的pEGFP-N3-aPPA2在COS7细胞中正常表达并检测出高的植酸酶活性。本研究提出的pEGFP-N3-aPPA2重组质粒构建和在哺乳类COS7细胞表达体系为植酸酶生产提供了新的技术线路。

A bacterial phytase cDNA was amplified with regular PCR technique and it contains 1 360 bp encoding 423 amino acids with protein molecular size approximately 48 kD. The cDNA fragment was sub-cloned into pEGFP-N3 plasmid DNA at the cloning site of BamH1/Pst1 . The recombinant pEGFP-aPPA2 plasmid DNA were transfected into mammalian COS7 cells. The recombinant plasmid was expressed in the mammalian cells and the fusion proteins exhibited high phytase activity. This study provides a new system to produce phytase with a potential for an application in related industry.