| 王伟杜美陈欢陆婕△.人胱硫醚β合成酶原核表达纯化及鉴定[J].,2011,11(5):830-833 |
| 人胱硫醚β合成酶原核表达纯化及鉴定 |
| Expression, Purification and Characterization of Recombinant HumanCBS in Escherichia coli |
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| DOI： |
| 中文关键词: 胱硫醚β合成酶 同型半胱氨酸 酶活力 原核表达 pET32a(+) |
| 英文关键词: CBS Hcy Enzymic activity Prokaryocyte expression pET32a(+) |
| 基金项目:湖北省自然科学基金（2009CDB076），华中科技大学自主创新研究基金（2010MS009 ) |
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| 中文摘要: |
| 目的：构建人胱硫醚β 合成酶(human cystathionine β-synthase, hCBS)基因原核表达载体，在E. coli BL21(DE3)中表达，并进
行纯化和酶活性检测。方法：以胰腺细胞cDNA 文库为模板，采用聚合酶链式反应(PCR)扩增hCBS 基因蛋白编码区的全序列，克
隆入原核表达载体pET32a(+)，构建重组质粒pET32a(+)-hCBS。经限制性内切酶双酶切及DNA 序列分析鉴定目的基因后与人
CBS 基因(基因bank 号：BT007154.1)完全一致，转入E. coli BL21(DE3)中，由IPTG 诱导表达融合蛋白。结果：经SDS-PAGE、
Western blot 分析，证明诱导表达的蛋白为重组人CBS(rhCBS)。再由Ni-NTA 树脂亲和层析，并脱盐冷冻干燥后获得重组rhCBS
(约19 mg/L 培养物)，并测得其比活力约为57 kU/g。结论：成功地表达纯化出具有功能活性的重组蛋白rhCBS，为进一步研究该酶
的相互作用蛋白以及其在生物学和临床科学的作用奠定了基础。 |
| 英文摘要: |
| Objective: To construct the human cystathionine β-synthase express vector, and to investigate the mechanism and
pharmaceutical function. Methods: The gene encoding hCBS was amplified by RT-PCR from human pancreatic cell cDNA library, then
was inserted into the expression vector pET32a (+) to construct the pET32a (+)-CBS. The recombinant human CBS (rhCBS) was
expressed in Escherichia coli (E .coli). By restriction enzyme digestion and DNA sequencing analysis, the right recombinant vector was
transformed into E. coli BL21 (DE3), and rhCBS was expressed by IPTG induction and purified by Ni-NTA affinity chromatography,
determined by SDS-PAGE and Western blotting analysis. Results: The gene inserted into pET32a (+) was exactly as the same as hCBS
gene . A total of 19 mg of high purity (over 98%) rhCBS was obtained from 1 L culture. Conclusions: The rhCBS with functional activity
was successfully expressed, and laid the foundations for further study of its interacted protein and its roles in biology and clinical science
research. |
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