| pET28a-TAT-LacZ重组子的构建 |
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| 引用本文： | 严世荣,龚坚,严洁,邱云城.pET28a-TAT-LacZ重组子的构建[J].遗传,2003,25(2):141-144. |
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| 作者姓名： | 严世荣 龚坚 严洁 邱云城 |
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| 作者单位： | 1.郧阳医学院附属十堰市人民医院检验部，湖北 十堰 442000；2.郧阳医学院生物化学教研室,湖北 十堰 442000;3.郧阳医学院附属太和医院神经内科，湖北 十堰 4420001. |
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| 基金项目： | 湖北省教育厅青年基金(2000A43006)资助 |
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| 摘 要： | 为了构建高表达pET28a-TAT-LacZ重组子，观察表达的融合蛋白TAT-β-Gal能否穿过生物膜，使用人工合成编码TAT蛋白转导区的DNA片段，插入载体pET28a组氨酸编码区后再连接lacZ基因，组成pET28a-TAT-LacZ重组表达子，转化大肠杆菌，利用组氨酸亲和层析柱纯化TAT-β-Gal融合蛋白，将融合蛋白加入培养的平滑肌细胞。得到高度纯化的、有活性的TAT-β-Gal融合蛋白， TAT-β-Gal在短时间内进入体外培养平滑肌细胞，成功地构建了高表达pET28a-TAT-LacZ重组子，并在体外培养的细胞中证实TAT-β-Gal融合蛋白穿透生物膜的能力，为肽类、生物大分子药物进入组织细胞内发挥治疗作用提供了理论基础。
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| 关 键 词： | 平滑肌细胞 TAT-β-Gal融合蛋白 TAT蛋白转导区 pET28a质粒 |
| 文章编号： | 0253-9772(2003)02-0141-04 |
| 修稿时间： | 2002年4月17日 |
Constructing the recombinant of pET28a-TAT-LacZ |
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| Shi-Rong Yan,Jian Gong,Jie Yan,Yun-Cheng Qiu.Constructing the recombinant of pET28a-TAT-LacZ[J].Hereditas,2003,25(2):141-144. |
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| Authors: | Shi-Rong Yan Jian Gong Jie Yan Yun-Cheng Qiu |
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| Institution: | Department of Clinical laboratory,Shiyan Peoples Hospital of Yunyang medical college,Shiyan,Hubei 442000,China;2.Department of Biochemistry Yunyang Medical College,Shiyan,Hubei 442000,China；3.Taihe Hospital of Yunyang Medical College,Shiyan,Hubei 442000,China |
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| Abstract: | A synthesized double-stranded oligomeric nucleotide encoding 11-amino acid TAT protein transduction domain3 was inserted into pET28a vector after 6 histidine coding sequence, LacZ gene from pcDNA4/Myc-His/LacZ was digested by EcoR I and Hind III, then cloned into pET28a-TAT. The highly expressed TAT-beta-galactosidase was purified by affinity chromatography. TAT-beta-Gal fusion protein can across vascular smooth muscle cells in vitro. This result open new possibility for direct delivery of protein into tissues for therapy. |
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