Construction of A New Phytase Gene Expression Vector and Its Over Expression in E.coli
phyA基因新型表达质粒的构建及其在大肠杆菌中的高效表达

This research amplified the phyA gene with the designed and synthesized specific primers for the phyA gene full-length coding sequence. The phyA gene was from Aspergillus niger F246 by the polymerase chain reaction (PCR), Which is selected and identified in our laboratory. After sequncing and amino-analyzing the coding sequence, it was confirmed that the construction of cloning vector was succeeded. The phyA gene fragment was recovered from the pMD18T-phyA and ligated with prokaryotic expression vector pET30a to construct the recombinant expression plasmid pET30a -phyA. It was expressed with IPTG induction in E. coli for high efficiency. A new protein band with apparent molecular weight 50kDa was detected in the lysate of the transformed cell expression by using SDS-PAGE, the amount of the soluble fusion protein was 36.62% of large intestine bacillus soluble protein of transformed cells, estimated by absorbance scanning of SDS-PAGE and protein quantitation. It' s phytase activity was four times over natural phyase. So this shows that the phyA gene has natural functions and is the base of the study on obtaining large and high active phytase and developing the new microbial ecologicalagent.