鸡滑液支原体乳酸脱氢酶的克隆表达及其表达产物活性研究

CLONING AND EXPRESSION OF LACTATE DEHYDROGENASE GENE OF MYCOPLASMA SYNOVIAE AND DETERMINATION OF ENZYMATIC ACTIVITY OF THE RECOMBINANT PROTEIN

根据GenBank中鸡滑液支原体(Mycoplasma synoviae,MS)MS53株的乳酸脱氢酶序列设计一对特异性上下游引物,通过PCR扩增获得MS WVU1853菌株的乳酸脱氢酶基因并将其克隆至pGEM-T Easy载体,测序正确并成功完成点突变后连接表达菌pET-28a(+),将重组表达质粒pET-28a-ldh转化表达菌BL21(DE3)。经IPTG诱导成功表达蛋白rMSLDH并测定其酶促活性。结果显示,重组表达的rMSLDH具有较强酶活性;最适反应温度40℃,最适pH7.5;Al2+和Ba2+可增加酶活性;Cr3+作为该酶的辅因子,在一定浓度内能有效的促进酶促反应进行;Cu2+作用下酶几乎失活,Fe3+、Mn2+、Ni2+、Zn2+对酶产生不同程度的抑制作用;其米氏常数(Km)为0.365 mmol/L,最大反应速率(Vm)为0.98μmol(L●min)。本研究结果为更好地研究鸡滑液支原体乳酸脱氢酶的生物学功能提供了参考。

In the present study, a pair of specific primers of lactate dehydrogenase (LDH) were designed according to the sequence of Mycoplasma synoviae MS 53 strain in GenBank. Then, the LDH gene of MS WVU1853 strain was amplified in PCR and cloned into pGEM-T Easy. After sequencing and spot mutation, the LDH gene was cloned into prokaryotic expression plasmid pET-28a (+). The recombinant plasmid pET-28a-LDH was transformed into BL21 (DE3). The expression of rLDH was induced with IPTG and its enzymatic activity was detected. The results showed that rLDH possessed high activity. The optimal reaction conditions were determined to be 40℃and pH 7.5. Its enzymatic activity was stimulated with addition of Ba2+, Al3+and Cr3+but inhabited with Fe3+, Mn2+, Ni2+and Zn2+. The michaelis constant (Km) of LDH was 0.365 mmol/L and the maximum reaction velocity (Vm) was 0.98μmol(Lmin).