| Reteplase（K）原核分泌表达载体的构建及其初步表达 |
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| 引用本文： | 罗惠霞,李敏,王玉炯. Reteplase（K）原核分泌表达载体的构建及其初步表达[J]. 生物学杂志, 2010, 27(5): 7-10,30. DOI: 10.3969/j.issn.1008-9632.2010.05.007 |
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| 作者姓名： | 罗惠霞 李敏 王玉炯 |
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| 作者单位： | 西部特色生物资源保护与利用教育部院重点实验室,宁夏银川,750021 |
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| 基金项目： | 宁夏自然科学基金资助 |
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| 摘 要： | 在原核表达载体pErA（K）的基础上,构建新型原核分泌表达载体pBrA（K）,并实现其在原核表达系统中的表达。用限制性内切酶NcoI,XhoⅠ同时双酶切获得目的基因rPA（K）后,以T4连接酶将其连接到pET-20b（＋）上,构建新型原核分泌表达载体pBrA（K）并实现了在大肠杆菌中表达,经纤维蛋白平板检测存在于重组菌株培养液上清中的分泌蛋白有溶纤活性。ELISA结果显示表达产物与抗t-PA抗体呈现特异性阳性反应,进一步参考标准曲线得出样品中rPA（K）的平均含量为51 ng/mL。实验结果证明新型原核分泌表达载体pBrA（K）构建成功,并实现了其在原核表达系统中的表达,为新型rPA（K）的表达优化及后续的纯化研究工作奠定了基础。
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| 关 键 词： | 组织型纤溶酶激活剂（t-PA） rPA（K） 原核分泌表达 |
The construction and expression of Reteplase(K)prokaryotic secretory expressive vector |
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| LUO Hui-xia,LI Min,WANG Yu-jiong. The construction and expression of Reteplase(K)prokaryotic secretory expressive vector[J]. Journal of Biology, 2010, 27(5): 7-10,30. DOI: 10.3969/j.issn.1008-9632.2010.05.007 |
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| Authors: | LUO Hui-xia LI Min WANG Yu-jiong |
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| Affiliation: | (Key laboratory of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China,Yinchuan 750021,China) |
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| Abstract: | On the base of pErA（K）,pBrA（K） would be constructed and the expression in the prokaryotic secretory system would be achieved.with Nco I and Xho I used by T4 ligase.Through mFAPA,the secretory protein in supernatant had the fibrinolysis activity.ELISA showed that the expressive product had specific positive response with anti-t-PA antibody and its average content was 51 ng/mL.The results showed that pBrA（K） had been constructed successfully,and the expression in the prokaryotic secretory system was achieved. |
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| Keywords: | rPA(K) |
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