牛支原体二氢硫辛酸脱氢酶基因的克隆表达及表达产物酶比活性的测定

Cloning and expression of dihydrolipoamide dehydrogenase gene from Mycoplasma bovis and determination of enzymatic activity of the recombinant protein

参照GenBank中牛支原体PG45株二氢硫辛酸脱氢酶基因(pdhD)的序列设计特异性引物,应用PCR扩增牛支原体武威株的pdhD基因,然后将其克隆至pMD19-T。在完成测序及点突变的基础上构建重组表达质粒pET-28a(+)-pdhD,转化大肠杆菌BL21(DE3),经IPTG诱导后纯化表达产物His-DLD,并对其酶促反应活性进行测定。结果显示,表达产物在底物NADH存在条件下,能将硫辛酰胺催化生成二氢硫辛酰胺,且其最适酶促反应温度为25℃、pH为7.5;其米氏常数Km(NADH)为9.72μmol/L,最大反应速率为25.6μmol/(L·min)。上述结果为研究二氢硫辛酸脱氢酶的生物学功能奠定了基础。

According to the sequence of dihydrolipoamide dehydrogenase（pdhD）gene of Mycoplasma bovis PG45 strain in GenBank,apair of specific primers was designed.Then,the pdhD gene of M.bovis Wuwei strain was amplified by PCR and cloned into pMD19-T vector.After sequencing and point mutation,the pdhD gene was cloned into the prokaryotic expression plasmid pET-28a（＋）.The recombinant plasmid pET-28a（＋）-pdhD was transformed into Escherichia coli BL21（DE3）.The expression of recombinant protein His-DLD was induced with IPTG and its enzymatic activity was determined.In result,the His-DLD could catalyze lipoamide to produce dihydrolipoamide,and the optimal reaction condition was as follows：temperature was at 25 ℃ and pH value was 7.5.The Michaelis constant（Km）of His-DLD was9.72μmol/L and the maximum of reaction velocity（Vmax）was 25.6μmol/（L· min）.In conclusion,the above-mentioned results laid the solid foundation for further study on biological functions of DLD.