phyA基因克隆及其在大肠杆菌中的高效表达

Cloning of a phyA Gene and Its Over Expression in E.coli.

利用自行筛选、鉴定的黑曲霉F246,根据植酸酶(phyA)基因成熟肽编码序列设计引物,直接PCR扩增phyA,经酶切分析、DNA测序分析证实phyA基因克隆成功。从pMD18T-phyA克隆中获得phyA编码序列,将其与pET30a+质粒连接,构建pET30a+-phyA重组质粒,并在大肠杆菌中获得了高效表达。重组质粒经IPTG诱导表达,SDS-PAGE特异区带分子量为50 kDa,此重组蛋白占大肠杆菌可溶性蛋白的36.62%,酶活性较天然植酸酶高8倍以上。本研究为大量获得高活性植酸酶以及开发新型微生态制剂奠定了基础。

This research amplified the phyA gene with the designed and synthesized primers specific 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 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 50 kDa was detected in the lysate of the transformed cell by using SDS-PAGE. The amount of the soluble fusion protein was about 40% of large intestine bacillus soluble protein of transformed cells, estimated by absorbance scanning of SDS-PAGE and protein quantitation. It＇s phytase activity was eight times over the natural phyase. So this research provides the basis of the study on obtaining large and high active phytase and developmant of the new microbial ecologicalagent.