| Ssp DnaB intein大肠杆菌中介导FVIII重链和轻链的连接 |
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| 引用本文： | 朱甫祥,刘泽隆,屈慧鸽,辛晓林,董洪新,刘相钦. Ssp DnaB intein大肠杆菌中介导FVIII重链和轻链的连接[J]. 生物工程学报, 2009, 25(7): 1101-1106 |
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| 作者姓名： | 朱甫祥 刘泽隆 屈慧鸽 辛晓林 董洪新 刘相钦 |
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| 作者单位： | 1. 鲁东大学生命科学学院,烟台,264025
2. Department of Biochemistry and Molecular Biology,Dalhousie University,Halifax B3H 4H7,Canada |
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| 基金项目： | 山东省自然科学基金(No.Y2005D14);;烟台市科技计划项目(No.2008152);;教育部留学回国人员科研启动基金;;鲁东大学科研启动经费项目资助~~ |
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| 摘 要： | 研究利用intein的蛋白质反式剪接功能在大肠杆菌中对凝血VIII因子(FVIII)重链和轻链的连接作用,将B结构域大部分缺失型FVIII(BDD-FVIII)于满足剪接所需的保守性氨基酸Ser1657前断裂为重链和轻链,分别与split mini Ssp DnaB intein的106个氨基酸的N端(Int-N)和48个氨基酸的C端(Int-C)融合,构建到原核表达载体pBV220。诱导表达后SDS-PAGE分析可见预期大小的BDD-FVIII蛋白条带,Western blotting用FVIII特异性抗体证明其为剪接所产生的BDD-FVIII蛋白,表明intein可有效连接BDD-FVIII的重链和轻链。为进一步甲型血友病基因治疗研究应用intein以双腺相关病毒载体(AAV)携带FVIII基因,克服单个AAV载体的容量限制提供了依据。
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| 关 键 词： | Intein 蛋白质反式剪接 凝血VIII因子 |
| 收稿时间： | 2009-03-23 |
Ssp DnaB intein-mediated ligation of heavy and light chains of coagulation factor VIII in Escherichia coli |
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| Fuxiang Zhu,Zelong Liu,Huige Qu,Xiaolin Xin,Hongxin Dong and Xiangqin Liu. Ssp DnaB intein-mediated ligation of heavy and light chains of coagulation factor VIII in Escherichia coli[J]. Chinese journal of biotechnology, 2009, 25(7): 1101-1106 |
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| Authors: | Fuxiang Zhu Zelong Liu Huige Qu Xiaolin Xin Hongxin Dong Xiangqin Liu |
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| Affiliation: | Life Science College of Ludong University, Yantai 264025, China;Life Science College of Ludong University, Yantai 264025, China;Life Science College of Ludong University, Yantai 264025, China;Life Science College of Ludong University, Yantai 264025, China;Life Science College of Ludong University, Yantai 264025, China;Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax B3H 4H7, Canada |
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| Abstract: | We studied the ligation of coagulation factor VIII heavy and light chains in Escherichia coli by utilizing the intein-mediated protein trans-splicing. A B-domain deleted factor VIII (BDD-FVIII) gene was broken into two halves of heavy and light chains before Ser1657 which meets the splicing required conserved residue and then fused to 106 and 48 amino acid-containing N-part termed Int-N and C-part termed Int-C coding sequences of split mini Ssp DnaB intein respectively. These two fusion genes were construct... |
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| Keywords: | intein protein trans-splicing coagulation factor VIII |
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