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当前位置: 首页 > 文章 > 滑液支原体烯醇化酶基因的克隆表达及其表达产物活性的测定 中国兽医科学 2013 (4) 416-420
Position: Home > Articles > Cloning and expression of enolase gene of Mycoplasma synoviae and determination of activity of the recombinant protein Chinese Veterinary Science 2013 (4) 416-420

滑液支原体烯醇化酶基因的克隆表达及其表达产物活性的测定

作  者：
郭小琴;包世俊;谭磊;何随彬;张凡庆
单  位：
甘肃农业大学动物医学院陇西县动物疫病预防控制中心;甘肃农业大学动物医学院;南京农业大学动物医学院中国农业科学院上海兽医研究所;中国农业科学院上海兽医研究所;甘肃农业大学动物医学院中国农业科学院上海兽医研究所
关键词：
滑液支原体;烯醇化酶基因;克隆;表达;酶活性
摘  要：
参照GenBank中滑液支原体P53株序列设计1对特异性引物,通过PCR扩增获得滑液支原体WVU1853菌株烯醇化酶基因并将其克隆至pGEM-T Easy,测序并完成点突变后构建重组表达质粒pET-28a-eno,转化大肠杆菌BL21(DE3),经IPTG诱导表达后纯化表达产物并测定其酶促活性。结果显示,表达产物具有水解酶活性,最适pH为7.5,最适温度为50℃,Mg2+可作为酶活性辅因子,相同浓度的Zn2+可提高酶活性,而Mn3+、Al 2+、Ni 2+、Ba2+和Li+对酶活性均有不同程度的抑制作用;其米氏常数(km)和最大反应速率(Vmax)分别为1.1×10-3 mol/L和0.739μmol/(L.min)。
译  名：
Cloning and expression of enolase gene of Mycoplasma synoviae and determination of activity of the recombinant protein
作  者：
GUO Xiao-qin1,BAO Shi-jun1,TAN Lei2,HE Sui-bin2,ZHANG Fan-qing2,QIU Xu-sheng2,SONG Cui-ping2,DING Chan2(1.College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;2.Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China)
关键词：
Mycoplasma synoviae;enolase gene;cloning;expression;enzymatic activity
摘  要：
According to the sequence of Mycoplasma synoviae P53 in GenBank,a pair of primers was designed and then the enolase gene of Mycoplasma synoviae WUV 1853 was amplified by PCR.After sequencing and point mutation,the gene was cloned into prokaryotic expression plasmid pET-28a(+).The recombinant plasmid pET-28a-eno was transformed into E.coli BL21(DE3).The expression was induced by IPTG and the fusion protein His-Eno was purified and its enzymatic activity was determined.The results showed that the recombinant enolase has the hydrolase activity,the optimal reaction condition of pH was 7.5 and temperature was 50 ℃.Its activity can be stimulated by Mg2+ and Zn2+,but can be inhibited by Mn3 +,Al2+,Ni2 +,Ba2 + and Li+.Michaelis constant(km) and maximum reaction velocity(Vmax) of the reaction was determined to be 1.1×10-3 mol/L and 0.739 μmol/(L·min) from the Lineweaver-Burk plot,respectively.

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