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| 人可溶性TRAIL cDNA的设计、合成与克隆 | |
| 引用本文： | 杨文理,陈守春,陈毅荣,覃扬.人可溶性TRAIL cDNA的设计、合成与克隆[J].泸州医学院学报,2005,28(5):397-401. |
| 作者姓名： | 杨文理 陈守春 陈毅荣 覃扬 |
| 作者单位： | 1. 泸州医学院生物化学教研室,四川泸州,646000 2. 地奥制药集团新药研究所 3. 四川大学基础与法医学院生物化学与分子生物学教研室 |
| 摘 要： | 目的:合成可溶性的肿瘤坏死因子相关的凋亡诱导配体(TRAIL)的cDNA,并构建其原核表达载体.方法:根据大肠杆菌密码子偏好性和表达载体多克隆位点限制性内切酶要求,设计基因拼接引物,合成TRAIL基因胞膜外段双链cDNA序列,将其连接于克隆载体pMD18-T中;将酶切、纯化的TRAIL基因与经相同处理的载体PSC相连接,转化感受态大肠杆菌JM109,筛选阳性重组子.结果:成功获得了包含TRAIL基因胞膜外段的双链cDNA序列,并成功构建了TRAIL胞膜外段原核表达载体PSC/TRAIL 111-281.结论:采用PCR的方法,实现合成寡核苷酸片段的一次性组装,方便了进一步的克隆. |
| 关 键 词： | 肿瘤坏死因子相关的凋亡诱导配体 基因合成 聚合酶链式反应 |
| 文章编号： | 1000-2669(2005)5-0397-05 |
| 修稿时间： | 2005年6月7日 |
THE DESIGN, CHEMICAL SYNTHESIS AND CLONING OF THE HUMAN TNF-RELATED APOPTOSIS-INDUCING LIGAND cDNA |
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| Yang Wenli,et al.THE DESIGN, CHEMICAL SYNTHESIS AND CLONING OF THE HUMAN TNF-RELATED APOPTOSIS-INDUCING LIGAND cDNA[J].Journal of Luzhou Medical College,2005,28(5):397-401. | |
| Authors: | Yang Wenli |
| Institution: | Yang Wenli,et alDepartment of Biochemistry and Molecular Biology,Luzhou Medical College |
| Abstract: | Objective: To synthesize the soluble TNF-related apoptosis-inducing ligand (TRAIL)cDNA and construct its prokaryotic expression vector. Methods: According to the high-usage codons in escherichia coli and the multiple cloning site of expression vector PSC, the extracellular region of TRAIL gene was designed and synthesized, which was then cloned into pMD18-T vector. After pMD18-T/TRAIL and PSC were digested, the target fragment purified was linked into the expression vector PSC, which was transferred into the competent cell JM109, and positive recombinant was screened. Results: The extracellular region of TRAIL gene was obtained successfully,and a prokaryotic expression vector PSC/TRAIL was constructed successfully. Conclusion: The synthetic oligonucleotide fragments were assembled in only one step by a PCR approach, which is convenient to be further cloneed. |
| Keywords: | TRAIL Gene chemical synthesis PCR |
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