| 柏 学,李彩虹,佟红娜,胡克平,高 海.人源酪蛋白激酶Ⅱ催化亚基的构建及表达纯化[J].,2020,(7):1201-1205 |
| 人源酪蛋白激酶Ⅱ催化亚基的构建及表达纯化 |
| Construction, Expression and Purification of Human Casein Kinase II Catalytic Subunit |
| 投稿时间：2019-10-22 修订日期：2019-11-16 |
| DOI：10.13241/j.cnki.pmb.2020.07.001 |
| 中文关键词: 原核表达 蛋白纯化 酪蛋白激酶Ⅱ csnk2a1 csnk2a2 |
| 英文关键词: Prokaryotic expression Protein purification Casein kinase 2 Csnk2a1 Csnk2a2 |
| 基金项目:中国医学科学院医学与健康科技创新工程项目（CIFMS 2016-12M-1-002） |
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| 中文摘要: |
| 摘要 目的：构建人源CK2催化亚基pET-28a(+)-hcsnk2a1和pET-28a(+)-hcsnk2a2重组质粒，并进行原核表达纯化得到高纯度融合蛋白，为进一步开展CK2生理病理机制研究以及CK2作为肿瘤抑制靶点相关抑制剂的筛选和评价提供实验基础。方法：利用合成的人源csnk2a1和csnk2a2目的基因片段，将酶切连接得到的重组质粒经测序验证后，进行大肠杆菌感受态BL21(DE3) / Transetta(DE3)转化。使用合适浓度IPTG诱导融合蛋白的表达以得到可溶性的融合蛋白，并应用AKTA avant蛋白纯化仪和Ni2+-NTA预装柱进行纯化，蛋白纯度最后经SDS-PAGE胶分离后考马斯亮蓝染色和Western Blot检测鉴定。结果：测序结果表明pET-28a(+)-hcsnk2a1和pET-28a(+)-hcsnk2a2质粒均成功被构建；经转化诱导表达后，成功纯化得到相对分子量为42KD的his-hcsnk2a1和38KD的his-hcsnk2a2目的融合蛋白。结论：首次成功构建得到pET-28a(+)-hcsnk2a1和pET-28a(+)-hcsnk2a2质粒；并表达纯化得到高浓度和高纯度的his-hcsnk2a1和his-hcsnk2a2融合蛋白，为后期的蛋白功能研究和抑制剂筛选评价提供了基础。 |
| 英文摘要: |
| ABSTRACT Objective: Construction of human CK2 catalytic subunits pET-28a(+)-hcsnk2a1 and pET-28a(+)-hcsnk2a2 plasmids; prokaryotic expression and purification of high purity protein. To further study the physiological and pathological mechanism of CK2; as well as screening and evaluation of CK2 as tumor suppressor target related inhibitors. Methods: Using the synthesized csnk2a1 and csnk2a2 gene fragments, the recombinant plasmid obtained by enzyme digestion and ligation. After verification by sequencing, the plasmids were transformed into Escherichia coli receptor BL21 (DE3) / Transetta (DE3). And the fusion protein was induced by IPTG of appropriate concentration to obtain soluble fusion protein. AKTA avant protein purifier and Ni2+-NTA pre-column were used for purification. The purity of the protein was identified by Coomassie brilliant blue staining and Western Blot after SDS-PAGE gel separation. Results: The sequencing results showed that both pET-28a(+)-hcsnk2a1 and pET-28a(+)-hcsnk2a2 plasmids were successfully constructed. Besides, the fusion proteins of his-hcsnk2a1 and his-hcsnk2a2 were successfully purified after induction and expression. Conclusion: The plasmids pET-28a(+)-hcsnk2a1 and pET-28a(+)- hcsnk2a2 were successfully constructed for the first time, and the high concentration and purity of his-hcsnk2a1（42KD） and his-hcsnk2a2（38KD） fusion proteins were obtained, which provided the basis for the later protein function research and inhibitor screening and evaluation. |
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