菠菜乙醇酸氧化酶基因的克隆及表达

采用RT-PCR技术从菠菜总RNA中分离扩增了乙醇酸氧化酶（GO）基因的cDNA序列，首先克隆到质粒pMD18T，进行了测序。然后将乙醇酸氧化酶的cDNA分别亚克隆至质粒pThioHisC、 pTIGTrx、pBV220和pET-2b(+)，分别转化大肠杆菌DH5α和BL21（DE3），并对重组乙醇酸氧化酶在大肠杆菌中的表达进行了研究。SDSPAGE和酶活分析表明，菠菜乙醇酸氧化酶在E.coli BL21 (DE3) （pTIGTrxGO）和E.coli BL21(DE3) （pET-22b(+)GO）里得到了高水平的表达，其中E.coli BL21(DE3) （pET-22b(+)GO）的乙醇酸氧化酶活性较高。

Cloning and Expression of Spinach Glycolate Oxidase in Escherichia coli

The cDNA coding spinach glycolate oxidase (GO) was amplified by RT PCR using the total RNA of spinach leaves as the template, and was cloned into cloning vector pMD18 T. After the DNA sequence was determined, the go gene was subcloned into E.coli expression vector pBV220, pET 22b(+), pTIG Trx and pThioHisC. SDS PAGE analysis revealed that the recombinant E.coli BL21 (DE3)(pTIG Trx GO) and E.coli BL21 (DE3)(pET 22b(+) GO) expressed the predicted 38kD glycolate oxidase, and the enzyme activity was also detected.