米根霉菌ldhL基因的克隆及其在大肠杆菌中的表达

Cloning and Expression of Rhizopus oryzae of ldhL Gene in Escherichia coli

摘要:

以米根霉菌基因组DNA为模板, 根据GenBank上已公布的米根霉L-乳酸脱氢酶基因(ldhL)序列设计特异性引物, PCR扩增得到963 bp的DNA片段, 经序列分析后将其亚克隆到原核表达载体pET30a上, 构建成重组质粒pET30a-ldhL。将pET30a-ldhL转化到BL21感受态细菌中, 经IPTG诱导表达后进行SDS-PAGE分析, 可见约43 kD的与预期大小一致的目的蛋白条带, 结果表明ldhL基因在大肠杆菌中进行了表达, 经酶活分析产物的酶活力为98 U/mL, 证明了表达产物具有预期的酶活性, 这为进一步研究利用乳清为发酵原料高产L-乳酸的米根霉基因工程菌株奠定了基础。

Abstract:

The ldhL-encoding gene was amplified from Rhizopus oryzae genome DNA using PCR technique, and the PCR product was approximately 1000 bp DNA segment. The PCR products were cloned into pMD18-T vector and identified by enzyme cutting analysis. The sequence results showed that ldhL gene is 963 bp. The DNA fragment of ldhL was subcloned into prokaryotic expression vector pET30a and the specific fusion protein with molecular weight 43 kD was expressed, the result showed that the cloned ldhL was expressed in BL21. The levels of ldhL activity expressed by E. coil BL21/ pET30a-ldhL were up to 98 U/mL. The results are expected to lay foundation for further studies on the gene engineering Rhizopus oryzae strain of high yield L(+)-lactic acid by fermentation of whey.