鸡滑液支原体磷酸甘油酸激酶的克隆表达及酶学活性测定

Cloning,Expression and Enzymatic Activity of Mycoplasma synoviae Phosphoglycerate Kinase

磷酸甘油酸激酶(PGK)是生物进行糖酵解的关键酶,目前未见关于鸡滑液支原体(MS)PGK的相关研究报道。本实验采用Overlap PCR方法对MS的pgk基因进行点突变扩增,然后连接至pET-28a(+)表达载体并在大肠杆菌BL21中表达,纯化获得重组MSPGK(rMSPGK)蛋白,然后对纯化后的rMSPGK蛋白的酶学活性进行测定,包括温度、pH及不同底物对酶活的影响,并计算其酶比活力、米氏常数及最大反应速率。结果显示:最终获得了纯化的rMSPGK蛋白,其相对分子质量约为45 kDa;酶学活性测定显示,rMSPGK蛋白的酶比活力为100.70 IU/mg,最适酶促温度为37℃,最适pH为7.5;双倒数法求得rMSPGK蛋白相对于3-PGA的最大反应速率Vmax为370.37μmol/(L?min),米氏常数Km值为3.88 mmol/L;相对于ATP的最大反应速率Vmax为357.14μmol/(L?min),Km值为0.25 mmol/L。综上,本实验成功地表达了MS的PGK蛋白,并获得了酶活力较高的rMSPGK蛋白,为进一步研究鸡滑液支原体代谢和致病机制奠定了基础。

Phosphoglycerate kinase(PGK)is an important glycolytic enzyme,which has not yet been studied in Mycoplasma synoviae(MS).In this study,the point-mutated MS pgk gene was obtained by overlaping PCR amplification and inserted to the pET28a(+)vector.The recombinant MSPGK(rMSPGK)protein was then expressed and purified from E.coli BL21.The enzymatic activity of purified rMSPGK,and its influencing factors,including temperature,pH and different substrates were evaluated.In addition,we calculated the specific enzyme activity,Michaelis constant(Km)and maximum reaction rate(Vmax)of purified rMSPGK.Finally,the purified rMSPGK protein was obtained with a relative molecular mass of about 45 kDa.The specific enzyme activity of the rMSPGK protein was determined to be 100.70 IU/mg and the optimum temperature and pH for enzyme reaction were 37℃and 7.5.By the double-reciprocal plots,the values of Vmax(3-PGA)and Km(3-PGA)were calculated to be 370.37μmol/(L·min)and 3.88 mmol/L and the Vmax(ATP)and the Km(ATP)were 357.14μmol/(L·min)and 0.25 mmol/L,respectively.In summary,the rMSPGK protein was successfully expressed and purified,which was then confirmed to have high phosphoglycerate kinase activity.This study laid a solid foundation for further research on the pathogenesis of MS infection.