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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  2017, Vol. 37 Issue (2): 81-87    DOI: 10.13523/j.cb.20170212
技术与方法     
循环酶法同型半胱氨酸检测关键酶CBS和CBL的开发及试剂盒研制初探
王笃强1,2, 沈云龙1, 廖远平1, 李德彬1, 刘虹均1, 陈帅1, 卢大儒2, 朱化星1
1. 吴江近岸蛋白质科技有限公司研发部 苏州 215222;
2. 复旦大学生命科学学院遗传工程国家重点实验室 上海 200433
Development of Two Recombinant Enzymes: Cystathionine β-synthase and Cystathionine β-lyase Vital for Enzymatic Cycling Homocysteine Assay and Preliminary Characterization of the Corresponding Assay Kit
WANG Du-qiang1,2, SHEN Yun-long1, LIAO Yuan-ping1, LI De-bin1, LIU Hong-jun1, CHEN Shuai1, LU Da-ru2, ZHU Hua-xing1
1. R&D Department, Novoprotein Scientific (Wujiang) Inc, Suzhou 215222, China;
2. State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China
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摘要：

以酿酒酵母基因组为模板通过PCR分别扩增胱硫醚β合成酶（cystathionine β-synthase，CBS）和胱硫醚β-裂解酶（cystathionine β-lyase，CBL）目的基因片段，经无缝克隆构建表达质粒并转化大肠杆菌菌株E. coli BL21（DE3）。经诱导表达和纯化后，重组蛋白的纯度均达到90%，回收率均达到80%，可溶表达量分别为26 mg/L和332 mg/L。经催化活性测定，CBS的单位酶活为15 U/mg，CBL的单位酶活为72 U/mg。在此基础上初步开发了循环酶法同型半胱氨酸（homocysteine，Hcy）检测试剂盒，实验结果证明该试剂盒的有效性和稳定性均符合体外诊断检测的要求，其检测性能与市售进口同类试剂盒基本一致。
关键词： -合成酶;  酶活测定;  胱硫醚β  胱硫醚β  -裂解酶;  同型半胱氨酸    
Abstract:

The target genes of cystathionine β-synthase (CBS) and cystathionine β-lyase (CBL) from Saccharomyces cerevisiae were amplified by PCR and cloned into the expression vector pET28a respectively. The resulting plasmids were then transformed to E.coli BL21(DE3) cells. After induction of the gene expression with IPTG and protein purification, the purities of recombinant CBS and CBL were both near 90% with a final yield of 80%. The soluble expression levels of CBS and CBL were 26 mg/L and 332 mg/L respectively, and the specific activities of purified enzymes were determined to be 15 U/mg and 72 U/mg. With utilization of recombinant CBS and CBL as two key components, an enzymatic cycling homocysteine (Hcy) assay kit was developed and characterized preliminarily, whose functionality and stability are believed to meet the requirements of in vitro diagnostic usage. In addition, functionality comparison of our Hcy assay kit with the imported product that has been sold on the market did not show any obvious difference.
Key words: Cystathionine β-lyase    Homocysteine    Cystathionine β-synthase    Enzymatic activity measurement
收稿日期: 2016-08-15 出版日期: 2017-02-25
ZTFLH:  Q819  
通讯作者: 朱化星, 卢大儒     E-mail: zhuhuaxing@novoprotein.com.cn;drlu@fudan.edu.cn
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引用本文:

王笃强, 沈云龙, 廖远平, 李德彬, 刘虹均, 陈帅, 卢大儒, 朱化星. 循环酶法同型半胱氨酸检测关键酶CBS和CBL的开发及试剂盒研制初探[J]. 中国生物工程杂志, 2017, 37(2): 81-87.

WANG Du-qiang, SHEN Yun-long, LIAO Yuan-ping, LI De-bin, LIU Hong-jun, CHEN Shuai, LU Da-ru, ZHU Hua-xing. Development of Two Recombinant Enzymes: Cystathionine β-synthase and Cystathionine β-lyase Vital for Enzymatic Cycling Homocysteine Assay and Preliminary Characterization of the Corresponding Assay Kit. China Biotechnology, 2017, 37(2): 81-87.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170212    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I2/81

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