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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (3): 16-23    DOI: 10.13523/j.cb.20180303
研究报告     
2型猪链球菌磷酸甘油酸激酶基因的克隆表达及酶活性测定 *
郭晓璐1,2,龚秀芳2,陈家锋2,丁晨曦2,胡丹2,潘秀珍2,王长军1,2*()
1 中国药科大学生命科学与技术学院 南京 211198
2 南京军区疾病预防控制中心 南京 210002
Gene Cloning, Expression and Identification of Phosphoglyceric Kinase of Streptococcus suis Serotype 2
Xiao-lu GUO1,2,Xiu-fang GONG2,Jia-feng CHEN2,Chen-xi DING2,Dan HU2,Xiu-zhen PAN2,Chang-jun WANG1,2*()
1 School of Life Science and Technology,China Pharmaceutical University,Nanjing 211198, China
2 Department of Epidemiology, Medicinal Research Institute, Nanjing Military Command, Nanjing　210002, China
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摘要： 目的

克隆表达2型猪链球菌中磷酸甘油酸激酶(PGK)并对其酶学特性进行测定。

 方法

采用PCR方法从05ZYH33基因组中扩增出pgk片段,构建重组表达质粒pET28a:pgk,经双酶切及测序验证正确的质粒转化入E.coli BL21(DE3)中进行IPTG诱导表达,重组PGK蛋白经SDS-PAGE和质谱鉴定并测定其酶学活性。

 结果

PGK在大肠杆菌中可溶性表达,纯化后得到约43kDa的重组PGK蛋白,其酶促反应最适温度为25℃,最适pH为7.5,2型猪链球菌PGK的酶活性为75U/ml,PGK相对于3-PGA的Km值为1.744mmol/L,Vmax为0.143mmol/(L·min),相对于ATP的Km值为2.266mmol/L,Vmax为0.318mmol/(L·min)。

 结论

利用原核表达系统成功地表达了2型猪链球菌中的PGK,并获得了活性较好的重组PGK,酶学检测发现纯化的PGK具有良好的体外活性,为进一步研究该病在2型猪链球菌致病及代谢机制奠定了基础。
关键词： 2型猪链球菌;  磷酸甘油酸激酶;  克隆表达;  酶活性    
Abstract: Objective:

To prokaryotically clone and express phosphoglycerate kinase(PGK) from Streptococcus suis serotype 2 and determine enzymatic properties of recombination protein.

 Methods:

pgk gene was amplified from the 05ZYH33 genome DNA by PCR and inserted into expression vector pET28a by double digestion. The combined prokaryotic expression plasmid pET28a:pgk was subsequently transformed into E.coli BL21 and induced by IPTG. The induction result was identified by SDS-PAGE and LC-MS/MS, the recombination PGK was then purified by Ni affinity chromatography and used for enzymatic activity measurement.

 Results:

PGK protein had a high and soluble expression and displayed a molecular weight about 43kDa in E.coli BL21. The enzymatic activity was then measured by using purified PGK. The enzymatic activity of recombination PGK protein was 75U/ml. The optimum temperature and pH was 25℃ and 7.5, respectively. Kinetic analyses with respect to 3-PGA as substrate gave a Km of 1.744mmol/L and ATP as substrate gave a Km of 2.266mmol/L.

 Conclusion:

The pgk gene has been successfully expressed by prokaryotic expression system and the purified recombinant PGK protein had a similar enzymatic activity other protein enzymes and showed the best enzymatic activity in optimum condition.
Key words: Streptococcus suis serotype 2    Phosphoglycerate kinase    Prokaryotic expression    Enzymatic activity
收稿日期: 2017-09-29 出版日期: 2018-04-04
ZTFLH:  Q819  
基金资助: 国家自然科学基金(81481920);军队后勤科研计划项目(AWS16J021)
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郭晓璐
龚秀芳
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丁晨曦
胡丹
潘秀珍
王长军

引用本文:

郭晓璐,龚秀芳,陈家锋,丁晨曦,胡丹,潘秀珍,王长军. 2型猪链球菌磷酸甘油酸激酶基因的克隆表达及酶活性测定 *[J]. 中国生物工程杂志, 2018, 38(3): 16-23.

Xiao-lu GUO,Xiu-fang GONG,Jia-feng CHEN,Chen-xi DING,Dan HU,Xiu-zhen PAN,Chang-jun WANG. Gene Cloning, Expression and Identification of Phosphoglyceric Kinase of Streptococcus suis Serotype 2. China Biotechnology, 2018, 38(3): 16-23.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180303    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I3/16

图1  pgk基因克隆及表达质粒构建
图2  SDS-PAGE鉴定PGK蛋白表达
图3  PGK质谱鉴定结果
图4  NADH光密度标准曲线
图5  重组PGK时间-吸光度曲线
图6  pH对重组PGK酶活的影响
图7  反应温度对PGK酶活性的影响
图8  3-PGA浓度对酶促反应速率的影响
图9  不同ATP浓度对酶促反应速率的影响
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