大鼠血红素加氧酶-1基因在乳酸乳球菌中的克隆与表达(英文)

Cloning and Expression of Rat Heme Oxygenase-1 Gene in Lactococcus lactis

采用RTPCR技术从大鼠脾总RNA中分离扩增HO1基因,将该基因克隆进pGEMTeasy质粒中,转化大肠杆菌DH5α,提取质粒,分析基因,酶切后与含有乳酸乳球菌启动子NisA的pSEC质粒连接,经电击转化,将重组质粒转入乳酸乳球菌NZ9000中,转化子在含有氯霉素的脑心浸液培养基上培养.用Nisin诱导HO1表达,SDSPAGE和Westernblot分析、鉴定表达产物,并且用分光光度法测定工程菌表达的HO1活性为0.45nmolmg(protein)-1h-1.

Total RNA was extracted from rat spleen tissue using RNA kit and heme oxygenase-1 （ HO-1 ） gene was amplified by RT-PCR. The sequence of HO-1 gene was identified by T/A clone and DNA sequencing. Then, cDNA of HO-1 was inserted into the N/s I/EcoR I site of the pSEC vector with PnisA promoter, resulting in the pSEC-HO-1 expressing vector. The vector was transformed into Lactococcus lactis NZ9000 by electroporation, and the expression of HO-1 gene induced by nisin was evaluated by SDS-PAGE and Western blot. In addition, the activity of HO-1 expressed by engineering L. lactis was 0.45 nmol mg（protein） -1 h 1 using spectrophotography. Fig 5, Ref 18